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Sample records for membrane fusion iii

  1. Membrane tension and membrane fusion.

    PubMed

    Kozlov, Michael M; Chernomordik, Leonid V

    2015-08-01

    Diverse cell biological processes that involve shaping and remodeling of cell membranes are regulated by membrane lateral tension. Here we focus on the role of tension in driving membrane fusion. We discuss the physics of membrane tension, forces that can generate the tension in plasma membrane of a cell, and the hypothesis that tension powers expansion of membrane fusion pores in late stages of cell-to-cell and exocytotic fusion. We propose that fusion pore expansion can require unusually large membrane tensions or, alternatively, low line tensions of the pore resulting from accumulation in the pore rim of membrane-bending proteins. Increase of the inter-membrane distance facilitates the reaction. PMID:26282924

  2. Viral membrane fusion.

    PubMed

    Harrison, Stephen C

    2015-05-01

    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.

  3. Viral membrane fusion

    SciTech Connect

    Harrison, Stephen C.

    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.

  4. The Domain I-Domain III Linker Plays an Important Role in the Fusogenic Conformational Change of the Alphavirus Membrane Fusion Protein▿

    PubMed Central

    Zheng, Yan; Sánchez-San Martín, Claudia; Qin, Zhao-ling; Kielian, Margaret

    2011-01-01

    The alphavirus Semliki Forest virus (SFV) infects cells through a low-pH-dependent membrane fusion reaction mediated by the virus fusion protein E1. Acidic pH initiates a series of E1 conformational changes that culminate in membrane fusion and include dissociation of the E1/E2 heterodimer, insertion of the E1 fusion loop into the target membrane, and refolding of E1 to a stable trimeric hairpin conformation. A highly conserved histidine (H3) on the E1 protein was previously shown to promote low-pH-dependent E1 refolding. An SFV mutant with an alanine substitution at this position (H3A) has a lower pH threshold and reduced efficiency of virus fusion and E1 trimer formation than wild-type SFV. Here we addressed the mechanism by which H3 promotes E1 refolding and membrane fusion. We identified E1 mutations that rescue the H3A defect. These revertants implicated a network of interactions that connect the domain I-domain III (DI-DIII) linker region with the E1 core trimer, including H3. In support of the importance of these interactions, mutation of residues in the network resulted in more acidic pH thresholds and reduced efficiencies of membrane fusion. In vitro studies of truncated E1 proteins demonstrated that the DI-DIII linker was required for production of a stable E1 core trimer on target membranes. Together, our results suggest a critical and previously unidentified role for the DI-DIII linker region during the low-pH-dependent refolding of E1 that drives membrane fusion. PMID:21543498

  5. Poxvirus entry and membrane fusion

    SciTech Connect

    Moss, Bernard . E-mail: bmoss@nih.gov

    2006-01-05

    The study of poxvirus entry and membrane fusion has been invigorated by new biochemical and microscopic findings that lead to the following conclusions: (1) the surface of the mature virion (MV), whether isolated from an infected cell or by disruption of the membrane wrapper of an extracellular virion, is comprised of a single lipid membrane embedded with non-glycosylated viral proteins; (2) the MV membrane fuses with the cell membrane, allowing the core to enter the cytoplasm and initiate gene expression; (3) fusion is mediated by a newly recognized group of viral protein components of the MV membrane, which are conserved in all members of the poxvirus family; (4) the latter MV entry/fusion proteins are required for cell to cell spread necessitating the disruption of the membrane wrapper of extracellular virions prior to fusion; and furthermore (5) the same group of MV entry/fusion proteins are required for virus-induced cell-cell fusion. Future research priorities include delineation of the roles of individual entry/fusion proteins and identification of cell receptors.

  6. A Model for Membrane Fusion

    NASA Astrophysics Data System (ADS)

    Ngatchou, Annita

    2010-01-01

    Pheochromocytoma is a tumor of the adrenal gland which originates from chromaffin cells and is characterized by the secretion of excessive amounts of neurotransmitter which lead to high blood pressure and palpitations. Pheochromocytoma contain membrane bound granules that store neurotransmitter. The release of these stored molecules into the extracellular space occurs by fusion of the granule membrane with the cell plasma membrane, a process called exocytosis. The molecular mechanism of this membrane fusion is not well understood. It is proposed that the so called SNARE proteins [1] are the pillar of vesicle fusion as their cleavage by clostridial toxin notably, Botulinum neurotoxin and Tetanus toxin abrogate the secretion of neurotransmitter [2]. Here, I describe how physical principles are applied to a biological cell to explore the role of the vesicle SNARE protein synaptobrevin-2 in easing granule fusion. The data presented here suggest a paradigm according to which the movement of the C-terminal of synaptobrevin-2 disrupts the lipid bilayer to form a fusion pore through which molecules can exit.

  7. Membrane fusion during phage lysis

    PubMed Central

    Berry, Joel; Kongari, Rohit; Cahill, Jesse; Young, Ry

    2015-01-01

    In general, phages cause lysis of the bacterial host to effect release of the progeny virions. Until recently, it was thought that degradation of the peptidoglycan (PG) was necessary and sufficient for osmotic bursting of the cell. Recently, we have shown that in Gram-negative hosts, phage lysis also requires the disruption of the outer membrane (OM). This is accomplished by spanins, which are phage-encoded proteins that connect the cytoplasmic membrane (inner membrane, IM) and the OM. The mechanism by which the spanins destroy the OM is unknown. Here we show that the spanins of the paradigm coliphage lambda mediate efficient membrane fusion. This supports the notion that the last step of lysis is the fusion of the IM and OM. Moreover, data are provided indicating that spanin-mediated fusion is regulated by the meshwork of the PG, thus coupling fusion to murein degradation by the phage endolysin. Because endolysin function requires the formation of μm-scale holes by the phage holin, the lysis pathway is seen to require dramatic dynamics on the part of the OM and IM, as well as destruction of the PG. PMID:25870259

  8. Molecular mechanism of mitochondrial membrane fusion.

    PubMed

    Griffin, Erik E; Detmer, Scott A; Chan, David C

    2006-01-01

    Mitochondrial fusion requires coordinated fusion of the outer and inner membranes. This process leads to exchange of contents, controls the shape of mitochondria, and is important for mitochondrial function. Two types of mitochondrial GTPases are essential for mitochondrial fusion. On the outer membrane, the fuzzy onions/mitofusin proteins form complexes in trans that mediate homotypic physical interactions between adjacent mitochondria and are likely directly involved in outer membrane fusion. Associated with the inner membrane, the OPA1 dynamin-family GTPase maintains membrane structure and is a good candidate for mediating inner membrane fusion. In yeast, Ugo1p binds to both of these GTPases to form a fusion complex, although a related protein has yet to be found in mammals. An understanding of the molecular mechanism of fusion may have implications for Charcot-Marie-Tooth subtype 2A and autosomal dominant optic atrophy, neurodegenerative diseases caused by mutations in Mfn2 and OPA1.

  9. Cell secretion and membrane fusion.

    PubMed

    Jena, Bhanu P

    2005-07-01

    Secretion occurs in all cells of multicellular organisms and involves the delivery of secretory products packaged in membrane-bound vesicles to the cell exterior. Specialized cells for neurotransmission, enzyme secretion or hormone release utilize a highly regulated secretory process. Secretory vesicles are transported to specific sites at the plasma membrane, where they dock and fuse to release their contents. Similar to other cellular processes, cell secretion is found to be highly regulated and a precisely orchestrated event. It has been demonstrated that membrane-bound secretory vesicles dock and fuse at porosomes, which are specialized supramolecular structures at the cell plasma membrane. Swelling of secretory vesicles results in a build-up of pressure, allowing expulsion of intravesicular contents. The extent of secretory vesicle swelling dictates the amount of intravesicular contents expelled during secretion. The discovery of the porosome, its isolation, its structure and dynamics at nm resolution and in real time, its biochemical composition and functional reconstitution into artificial lipid membrane, have been determined. The molecular mechanism of secretory vesicle fusion at the base of porosomes, and vesicle swelling, has also been resolved. These findings reveal the molecular mechanism of cell secretion.

  10. Fusion of Liposomes with Mitochondrial Inner Membranes

    NASA Astrophysics Data System (ADS)

    Schneider, Heinz; Lemasters, John J.; Hochli, Matthias; Hackenbrock, Charles R.

    1980-01-01

    A procedure is outlined for the fusion of mixed phospholipid liposomes (small unilamellar vesicles) with the mitochondrial inner membrane, which enriches the membrane lipid bilayer 30-700% in a controlled fashion. Fusion was initiated by manipulation of the pH of a mixture of freshly sonicated liposomes and the functional inner membrane/matrix fraction of rat liver mitochondria. During the pH fusion procedure, liposomes became closely apposed with and sequestered by the inner membranes as revealed by freeze-fracture electron microscopy. After the pH fusion procedure, a number of ultrastructural, compositional, and functional characteristics were found to be proportionally related: the membrane surface area increased; the lateral density distribution of intramembrane particles (integral proteins) in the plane of the membrane decreased whereas the particles remained random; the membrane became more buoyant; the ratio of membrane lipid phosphorus to total membrane protein increased; the ratio of membrane lipid phosphorus to heme a of cytochrome c oxidase increased; and the rate of electron transfer between some interacting membrane oxidoreduction proteins decreased. These data reveal that liposomal phospholipid was incorporated into the membrane bilayer (not simply adsorbed to the membrane surface) and that integral membrane proteins diffused freely into the laterally expanding bilayer. Furthermore, the data suggest that the rate of electron transfer may be limited by the rate of lateral diffusion of oxidoreduction components in the bilayer of the mitochondrial inner membrane.

  11. Self-contact elimination by membrane fusion.

    PubMed

    Sumida, Grant M; Yamada, Soichiro

    2013-11-19

    Mutual, homophilic cell-cell adhesion between epithelial cells is required for proper maintenance of epithelial barrier function. Whereas opposing membranes from neighboring cells rapidly assemble junctional complexes, self-contacting membranes curiously do not, suggesting that cells have the ability to prevent the maturation of self-junctions. Using a self-contact-inducing microfabricated substrate, we show that self-contacts of normal epithelial cells are rapidly eliminated by membrane fusion between two opposing plasma membranes of a single cell. This membrane fusion is most frequently observed in E-cadherin-expressing epithelial cells, but not in fibroblasts. The efficiency of self-contact elimination depends on extracellular calcium concentration and the level of E-cadherin, suggesting that E-cadherin, although not required, enhances membrane fusion efficiency by bringing opposing membranes into close apposition to one another. Additionally, Rho-associated protein kinase inhibition decreases self-contact-induced membrane fusion of epithelial cells, suggesting that this fusion may be mechanically regulated through the actin-myosin network. This self-contact-induced membrane fusion is a key elimination mechanism for unwanted self-junctions and may be a feature of cell self-recognition. PMID:24191042

  12. Mutation of the dengue virus type 2 envelope protein heparan sulfate binding sites or the domain III lateral ridge blocks replication in Vero cells prior to membrane fusion

    SciTech Connect

    Roehrig, John T.; Butrapet, Siritorn; Liss, Nathan M.; Bennett, Susan L.; Luy, Betty E.; Childers, Thomas; Boroughs, Karen L.; Stovall, Janae L.; Calvert, Amanda E.; Blair, Carol D.; Huang, Claire Y.-H.

    2013-07-05

    Using an infectious cDNA clone we engineered seven mutations in the putative heparan sulfate- and receptor-binding motifs of the envelope protein of dengue virus serotype 2, strain 16681. Four mutant viruses, KK122/123EE, E202K, G304K, and KKK305/307/310EEE, were recovered following transfection of C6/36 cells. A fifth mutant, KK291/295EE, was recovered from C6/36 cells with a compensatory E295V mutation. All mutants grew in and mediated fusion of virus-infected C6/36 cells, but three of the mutants, KK122/123EE, E202K, G304K, did not grow in Vero cells without further modification. Two Vero cell lethal mutants, KK291/295EV and KKK307/307/310EEE, failed to replicate in DC-SIGN-transformed Raji cells and did not react with monoclonal antibodies known to block DENV attachment to Vero cells. Additionally, both mutants were unable to initiate negative-strand vRNA synthesis in Vero cells by 72 h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion. - Highlights: • Heparan sulfate- and receptor-binding motifs of DENV2 envelope protein were mutated. • Four mutant viruses were isolated—all could fuse C6/36 cells. • Two of these mutants were lethal in Vero cells without further modification. • Lethal mutations were KK291/295EV and KKK305/307/310EEE. • Cell attachment was implicated as the replication block for both mutants.

  13. Deployment of membrane fusion protein domains during fusion.

    PubMed

    Bentz, J; Mittal, A

    2000-01-01

    It is clear that both viral and intracellular membrane fusion proteins contain a minimal set of domains which must be deployed at the appropriate time during the fusion process. An account of these domains and their functions is given here for the four best-described fusion systems: influenza HA, sendai virus F1, HIV gp120/41 and the neuronal SNARE core composed of synaptobrevin (syn), syntaxin (stx) and the N- and C-termini of SNAP25 (sn25), together with the Ca(2+)binding protein synaptotagmin (syt). Membrane fusion begins with the binding of the virion or vesicle to the target membrane via receptors. The committed step in influenza HA- mediated fusion begins with an aggregate of HAs (at least eight) with some of their HA2 N-termini, a.k.a. fusion peptides, embedded into the viral bilayer (Bentz, 2000 a). The hypothesis presented in Bentz (2000 b) is that the conformational change of HA to the extended coiled coil extracts the fusion peptides from the viral bilayer. When this extraction occurs from the center of the site of restricted lipid flow, it exposes acyl chains and parts of the HA transmembrane domains to the aqueous media, i.e. a hydrophobic defect is formed. This is the 'transition state' of the committed step of fusion. It is stabilized by a 'dam' of HAs, which are inhibited from diffusing away by the rest of the HAs in the aggregate and because that would initially expose more acyl chains to water. Recruitment of lipids from the apposed target membrane can heal this hydrophobic defect, initiating lipid mixing and fusion. The HA transmembrane domains are required to be part of the hydrophobic defect, because the HA aggregate must be closely packed enough to restrict lipid flow. This hypothesis provides a simple and direct coupling between the energy released by the formation of the coiled coil to the energy needed to create and stabilize the high energy intermediates of fusion. Several of these essential domains have been described for the viral fusion

  14. Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc

    PubMed Central

    Barriga, Gonzalo P.; Villalón-Letelier, Fernando; Márquez, Chantal L.; Bignon, Eduardo A.; Acuña, Rodrigo; Ross, Breyan H.; Monasterio, Octavio; Mardones, Gonzalo A.; Vidal, Simon E.; Tischler, Nicole D.

    2016-01-01

    Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses

  15. Inhibition of the Hantavirus Fusion Process by Predicted Domain III and Stem Peptides from Glycoprotein Gc.

    PubMed

    Barriga, Gonzalo P; Villalón-Letelier, Fernando; Márquez, Chantal L; Bignon, Eduardo A; Acuña, Rodrigo; Ross, Breyan H; Monasterio, Octavio; Mardones, Gonzalo A; Vidal, Simon E; Tischler, Nicole D

    2016-07-01

    Hantaviruses can cause hantavirus pulmonary syndrome or hemorrhagic fever with renal syndrome in humans. To enter cells, hantaviruses fuse their envelope membrane with host cell membranes. Previously, we have shown that the Gc envelope glycoprotein is the viral fusion protein sharing characteristics with class II fusion proteins. The ectodomain of class II fusion proteins is composed of three domains connected by a stem region to a transmembrane anchor in the viral envelope. These fusion proteins can be inhibited through exogenous fusion protein fragments spanning domain III (DIII) and the stem region. Such fragments are thought to interact with the core of the fusion protein trimer during the transition from its pre-fusion to its post-fusion conformation. Based on our previous homology model structure for Gc from Andes hantavirus (ANDV), here we predicted and generated recombinant DIII and stem peptides to test whether these fragments inhibit hantavirus membrane fusion and cell entry. Recombinant ANDV DIII was soluble, presented disulfide bridges and beta-sheet secondary structure, supporting the in silico model. Using DIII and the C-terminal part of the stem region, the infection of cells by ANDV was blocked up to 60% when fusion of ANDV occurred within the endosomal route, and up to 95% when fusion occurred with the plasma membrane. Furthermore, the fragments impaired ANDV glycoprotein-mediated cell-cell fusion, and cross-inhibited the fusion mediated by the glycoproteins from Puumala virus (PUUV). The Gc fragments interfered in ANDV cell entry by preventing membrane hemifusion and pore formation, retaining Gc in a non-resistant homotrimer stage, as described for DIII and stem peptide inhibitors of class II fusion proteins. Collectively, our results demonstrate that hantavirus Gc shares not only structural, but also mechanistic similarity with class II viral fusion proteins, and will hopefully help in developing novel therapeutic strategies against hantaviruses

  16. Cryomicroscopy provides structural snapshots of influenza virus membrane fusion.

    PubMed

    Calder, Lesley J; Rosenthal, Peter B

    2016-09-01

    The lipid-enveloped influenza virus enters host cells during infection by binding cell-surface receptors and, after receptor-mediated endocytosis, fusing with the membrane of the endosome and delivering the viral genome and transcription machinery into the host cell. These events are mediated by the hemagglutinin (HA) surface glycoprotein. At the low pH of the endosome, an irreversible conformational change in the HA, including the exposure of the hydrophobic fusion peptide, activates membrane fusion. Here we used electron cryomicroscopy and cryotomography to image the fusion of influenza virus with target membranes at low pH. We visualized structural intermediates of HA and their interactions with membranes during the course of membrane fusion as well as ultrastructural changes in the virus that accompany membrane fusion. Our observations are relevant to a wide range of protein-mediated membrane-fusion processes and demonstrate how dynamic membrane events may be studied by cryomicroscopy. PMID:27501535

  17. IM30 triggers membrane fusion in cyanobacteria and chloroplasts.

    PubMed

    Hennig, Raoul; Heidrich, Jennifer; Saur, Michael; Schmüser, Lars; Roeters, Steven J; Hellmann, Nadja; Woutersen, Sander; Bonn, Mischa; Weidner, Tobias; Markl, Jürgen; Schneider, Dirk

    2015-01-01

    The thylakoid membrane of chloroplasts and cyanobacteria is a unique internal membrane system harbouring the complexes of the photosynthetic electron transfer chain. Despite their apparent importance, little is known about the biogenesis and maintenance of thylakoid membranes. Although membrane fusion events are essential for the formation of thylakoid membranes, proteins involved in membrane fusion have yet to be identified in photosynthetic cells or organelles. Here we show that IM30, a conserved chloroplast and cyanobacterial protein of approximately 30 kDa binds as an oligomeric ring in a well-defined geometry specifically to membranes containing anionic lipids. Triggered by Mg(2+), membrane binding causes destabilization and eventually results in membrane fusion. We propose that IM30 establishes contacts between internal membrane sites and promotes fusion to enable regulated exchange of proteins and/or lipids in cyanobacteria and chloroplasts. PMID:25952141

  18. A Stable Prefusion Intermediate of the Alphavirus Fusion Protein Reveals Critical Features of Class II Membrane Fusion

    PubMed Central

    Martín, Claudia Sánchez-San; Sosa, Hernando

    2009-01-01

    Summary Alphaviruses infect cells via a low-pH-triggered membrane fusion reaction mediated by the class II virus fusion protein E1, an elongated molecule with three extramembrane domains (DI–III). E1 drives fusion by inserting its fusion peptide loop into the target membrane and refolding to a hairpin-like trimer in which DIII moves toward the target membrane and packs against the central trimer. Three-dimensional structures provide static pictures of prefusion and postfusion E1 but do not explain this transition. Using truncated forms of E1, we reconstituted a low-pH-dependent intermediate composed of trimers of DI/II. Unexpectedly, DI/II trimers were stable in the absence of DIII. Once formed at a low pH, DI/II trimers efficiently and specifically bound recombinant DIII through a pH-independent reaction. Even in the absence of DIII, DI/II trimers interacted to form hexagonal lattices and to cause membrane deformation and tubulation. These studies identify a prefusion intermediate in class II membrane fusion. PMID:19064260

  19. Stalk model of membrane fusion: solution of energy crisis.

    PubMed Central

    Kozlovsky, Yonathan; Kozlov, Michael M

    2002-01-01

    Membrane fusion proceeds via formation of intermediate nonbilayer structures. The stalk model of fusion intermediate is commonly recognized to account for the major phenomenology of the fusion process. However, in its current form, the stalk model poses a challenge. On one hand, it is able to describe qualitatively the modulation of the fusion reaction by the lipid composition of the membranes. On the other, it predicts very large values of the stalk energy, so that the related energy barrier for fusion cannot be overcome by membranes within a biologically reasonable span of time. We suggest a new structure for the fusion stalk, which resolves the energy crisis of the model. Our approach is based on a combined deformation of the stalk membrane including bending of the membrane surface and tilt of the hydrocarbon chains of lipid molecules. We demonstrate that the energy of the fusion stalk is a few times smaller than those predicted previously and the stalks are feasible in real systems. We account quantitatively for the experimental results on dependence of the fusion reaction on the lipid composition of different membrane monolayers. We analyze the dependence of the stalk energy on the distance between the fusing membranes and provide the experimentally testable predictions for the structural features of the stalk intermediates. PMID:11806930

  20. Transient domains induced by influenza haemagglutinin during membrane fusion.

    PubMed

    Blumenthal, R; Pak, C C; Raviv, Y; Krumbiegel, M; Bergelson, L D; Morris, S J; Lowy, R J

    1995-01-01

    During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

  1. Direct observation of intermediate states in model membrane fusion

    PubMed Central

    Keidel, Andrea; Bartsch, Tobias F.; Florin, Ernst-Ludwig

    2016-01-01

    We introduce a novel assay for membrane fusion of solid supported membranes on silica beads and on coverslips. Fusion of the lipid bilayers is induced by bringing an optically trapped bead in contact with the coverslip surface while observing the bead’s thermal motion with microsecond temporal and nanometer spatial resolution using a three-dimensional position detector. The probability of fusion is controlled by the membrane tension on the particle. We show that the progression of fusion can be monitored by changes in the three-dimensional position histograms of the bead and in its rate of diffusion. We were able to observe all fusion intermediates including transient fusion, formation of a stalk, hemifusion and the completion of a fusion pore. Fusion intermediates are characterized by axial but not lateral confinement of the motion of the bead and independently by the change of its rate of diffusion due to the additional drag from the stalk-like connection between the two membranes. The detailed information provided by this assay makes it ideally suited for studies of early events in pure lipid bilayer fusion or fusion assisted by fusogenic molecules. PMID:27029285

  2. Direct observation of intermediate states in model membrane fusion.

    PubMed

    Keidel, Andrea; Bartsch, Tobias F; Florin, Ernst-Ludwig

    2016-01-01

    We introduce a novel assay for membrane fusion of solid supported membranes on silica beads and on coverslips. Fusion of the lipid bilayers is induced by bringing an optically trapped bead in contact with the coverslip surface while observing the bead's thermal motion with microsecond temporal and nanometer spatial resolution using a three-dimensional position detector. The probability of fusion is controlled by the membrane tension on the particle. We show that the progression of fusion can be monitored by changes in the three-dimensional position histograms of the bead and in its rate of diffusion. We were able to observe all fusion intermediates including transient fusion, formation of a stalk, hemifusion and the completion of a fusion pore. Fusion intermediates are characterized by axial but not lateral confinement of the motion of the bead and independently by the change of its rate of diffusion due to the additional drag from the stalk-like connection between the two membranes. The detailed information provided by this assay makes it ideally suited for studies of early events in pure lipid bilayer fusion or fusion assisted by fusogenic molecules. PMID:27029285

  3. Imaging multiple intermediates of single-virus membrane fusion mediated by distinct fusion proteins.

    PubMed

    Joo, Kye-Il; Tai, April; Lee, Chi-Lin; Wong, Clement; Wang, Pin

    2010-09-01

    Membrane fusion plays an essential role in the entry of enveloped viruses into target cells. The merging of viral and target cell membranes is catalyzed by viral fusion proteins, which involves multiple sequential steps in the fusion process. However, the fusion mechanisms mediated by different fusion proteins involve multiple transient intermediates that have not been well characterized. Here, we report a synthetic virus platform that allows us to better understand the different fusion mechanisms driven by the diverse types fusion proteins. The platform consists of lentiviral particles coenveloped with a surface antibody, which serves as the binding protein, along with a fusion protein derived from either influenza virus (HAmu) or Sindbis virus (SINmu). By using a single virus tracking technique, we demonstrated that both HAmu- and SINmu-bearing viruses enter cells through clathrin-dependent endocytosis, but they required different endosomal trafficking routes to initiate viral fusion. Direct observation of single viral fusion events clearly showed that hemifusion mediated by SINmu upon exposure to low pH occurs faster than that mediated by HAmu. Monitoring sequential fusion processes by dual labeling the outer and inner leaflets of viral membranes also revealed that the SINmu-mediated hemifusion intermediate is relatively long-lived as compared with that mediated by HAmu. Taken together, we have demonstrated that the combination of this versatile viral platform with the techniques of single virus tracking can be a powerful tool for revealing molecular details of fusion mediated by various fusion proteins.

  4. Observations of membrane fusion in a liposome dispersion: the missing fusion intermediate?

    PubMed Central

    Foldvari, Marianna

    2015-01-01

    Early intermediate structures of liposome-liposome fusion events were captured by freeze-fracture electron microscopic (EM) technique. The images show the morphology of the fusion interface at several different stages of the fusion event. One of the intermediates was captured at a serendipitous stage of two vesicles’ membranes (both leaflets) merging and their contents starting to intermix clearly showing the fusion interface with a previously unseen fusion rim. From the morphological information a hypothetical sequence of the fusion event and corresponding lipid structural arrangements are described. PMID:26069726

  5. Recyclotron III, a recirculating plasma fusion system

    SciTech Connect

    Jarnagin, W.S.

    1987-01-27

    This patent describes a recyclotron nuclear fusion system comprising recyclotrons. Each recyclotron comprises cyclotron means for receiving and accelerating charged particles in spiral and work conservative pathways. An output means forms a beam from particles received from the cyclotron means; (i) the cyclotron means comprising (a) a channel shaped electromagnet having a pair of indented polefaces, oriented along an input axis and defining an input axis and defining an input magnetic well, (b) a pair of elongated linear electrodes centered along the input magnetic well arranged generally parallel to the input axis and having a gap therebetween, (c) tuned oscillator means connected to the electrodes for applying an oscillating electric potential thereto, (ii) the output means comprising (e) inverter means comprising an electromagnet having a polarity opposite that of the channel shaped electromagnet oriented contigously therealong for extracting fully accelerated particles from the cyclotron means, and (f) reinverter means comprising an electromagnet having a polarity the same as that of the channel shaped electromagnet for correcting the flightpath of the extracted particles.

  6. Membrane fusion by VAMP3 and plasma membrane t-SNAREs

    SciTech Connect

    Hu Chuan Hardee, Deborah; Minnear, Fred

    2007-09-10

    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 {alpha}-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.

  7. Calorimetric detection of influenza virus induced membrane fusion.

    PubMed

    Nebel, S; Bartoldus, I; Stegmann, T

    1995-05-01

    Membrane fusion induced by the hemagglutinin glycoprotein of influenza virus has been extensively characterized, but the mechanism whereby the protein achieves the merger of the viral and target membrane lipids remains enigmatic. Various lipid intermediate structures have been proposed, and the energies required for their formation predicted. Here, we have analyzed the enthalpies of fusion of influenza with liposomes by titration calorimetry. If a small sample of virus in a weak neutral pH buffer was added to an excess of liposomes at low pH, a two-component reaction was seen, composed of an exothermic reaction and a slower endothermic reaction. The exothermic reaction was the result of acid-base reactions between the neutral pH virus sample and low pH buffer and low-pH-induced changes in the virus. The endothermic reaction was not observed in the absence of liposomes and much reduced if acid-inactivated virus, which had lost its fusion but not its binding activity, was added to liposomes. The endothermic reaction was more temperature dependent than the exothermic reaction; its pH dependence corresponded with that of fusion and its enthalpy was higher if fusion was more extensive. These data indicate that most of the endothermic reaction was due to membrane fusion. The experimentally determined enthalpy of fusion, 0.6-0.7 kcal per mol of viral phospholipids, is much higher than expected on the basis of current theories about the formation of lipid intermediates during membrane fusion.

  8. The dengue virus type 2 envelope protein fusion peptide is essential for membrane fusion

    SciTech Connect

    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.

    2010-01-20

    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.

  9. Differential cholesterol binding by class II fusion proteins determines membrane fusion properties.

    PubMed

    Umashankar, M; Sánchez-San Martín, Claudia; Liao, Maofu; Reilly, Brigid; Guo, Alice; Taylor, Gwen; Kielian, Margaret

    2008-09-01

    The class II fusion proteins of the alphaviruses and flaviviruses mediate virus infection by driving the fusion of the virus membrane with that of the cell. These fusion proteins are triggered by low pH, and their structures are strikingly similar in both the prefusion dimer and the postfusion homotrimer conformations. Here we have compared cholesterol interactions during membrane fusion by these two groups of viruses. Using cholesterol-depleted insect cells, we showed that fusion and infection by the alphaviruses Semliki Forest virus (SFV) and Sindbis virus were strongly promoted by cholesterol, with similar sterol dependence in laboratory and field isolates and in viruses passaged in tissue culture. The E1 fusion protein from SFV bound cholesterol, as detected by labeling with photocholesterol and by cholesterol extraction studies. In contrast, fusion and infection by numerous strains of the flavivirus dengue virus (DV) and by yellow fever virus 17D were cholesterol independent, and the DV fusion protein did not show significant cholesterol binding. SFV E1 is the first virus fusion protein demonstrated to directly bind cholesterol. Taken together, our results reveal important functional differences conferred by the cholesterol-binding properties of class II fusion proteins.

  10. The effect of acute microgravity on mechanically-induced membrane damage and membrane-membrane fusion events

    NASA Technical Reports Server (NTRS)

    Clarke, M. S.; Vanderburg, C. R.; Feeback, D. L.; McIntire, L. V. (Principal Investigator)

    2001-01-01

    Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". Both membrane rupture and membrane resealing events mediated by membrane-membrane fusion characterize this response. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

  11. The Effect of Acute Microgravity on Mechanically-Induced Membrane Damage and Membrane-Membrane Fusion Events

    NASA Technical Reports Server (NTRS)

    Clarke, Mark, S. F.; Vanderburg, Charles R.; Feedback, Daniel L.

    2001-01-01

    Although it is unclear how a living cell senses gravitational forces there is no doubt that perturbation of the gravitational environment results in profound alterations in cellular function. In the present study, we have focused our attention on how acute microgravity exposure during parabolic flight affects the skeletal muscle cell plasma membrane (i.e. sarcolemma), with specific reference to a mechanically-reactive signaling mechanism known as mechanically-induced membrane disruption or "wounding". This response is characterized by both membrane rupture and membrane resealing events mediated by membrane-membrane fusion. We here present experimental evidence that acute microgravity exposure can inhibit membrane-membrane fusion events essential for the resealing of sarcolemmal wounds in individual human myoblasts. Additional evidence to support this contention comes from experimental studies that demonstrate acute microgravity exposure also inhibits secretagogue-stimulated intracellular vesicle fusion with the plasma membrane in HL-60 cells. Based on our own observations and those of other investigators in a variety of ground-based models of membrane wounding and membrane-membrane fusion, we suggest that the disruption in the membrane resealing process observed during acute microgravity is consistent with a microgravity-induced decrease in membrane order.

  12. Organelle acidification negatively regulates vacuole membrane fusion in vivo

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-07-01

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

  14. Expansion of the fusion stalk and its implication for biological membrane fusion

    PubMed Central

    Risselada, Herre Jelger; Bubnis, Gregory; Grubmüller, Helmut

    2014-01-01

    Over the past 20 years, it has been widely accepted that membrane fusion proceeds via a hemifusion step before opening of the productive fusion pore. An initial hourglass-shaped lipid structure, the fusion stalk, is formed between the adjacent membrane leaflets (cis leaflets). It remains controversial if and how fusion proteins drive the subsequent transition (expansion) of the stalk into a fusion pore. Here, we propose a comprehensive and consistent thermodynamic understanding in terms of the underlying free-energy landscape of stalk expansion. We illustrate how the underlying free energy landscape of stalk expansion and the concomitant pathway is altered by subtle differences in membrane environment, such as leaflet composition, asymmetry, and flexibility. Nonleaky stalk expansion (stalk widening) requires the formation of a critical trans-leaflet contact. The fusion machinery can mechanically enforce trans-leaflet contact formation either by directly enforcing the trans-leaflets in close proximity, or by (electrostatically) condensing the area of the cis leaflets. The rate of these fast fusion reactions may not be primarily limited by the energetics but by the forces that the fusion proteins are able to exert. PMID:25024174

  15. The Yeast Cell Fusion Protein Prm1p Requires Covalent Dimerization to Promote Membrane Fusion

    PubMed Central

    Engel, Alex; Aguilar, Pablo S.; Walter, Peter

    2010-01-01

    Prm1p is a multipass membrane protein that promotes plasma membrane fusion during yeast mating. The mechanism by which Prm1p and other putative regulators of developmentally controlled cell-cell fusion events facilitate membrane fusion has remained largely elusive. Here, we report that Prm1p forms covalently linked homodimers. Covalent Prm1p dimer formation occurs via intermolecular disulfide bonds of two cysteines, Cys-120 and Cys-545. PRM1 mutants in which these cysteines have been substituted are fusion defective. These PRM1 mutants are normally expressed, retain homotypic interaction and can traffic to the fusion zone. Because prm1-C120S and prm1-C545S mutants can form covalent dimers when coexpressed with wild-type PRM1, an intermolecular C120-C545 disulfide linkage is inferred. Cys-120 is adjacent to a highly conserved hydrophobic domain. Mutation of a charged residue within this hydrophobic domain abrogates formation of covalent dimers, trafficking to the fusion zone, and fusion-promoting activity. The importance of intermolecular disulfide bonding informs models regarding the mechanism of Prm1-mediated cell-cell fusion. PMID:20485669

  16. Resolving single membrane fusion events on planar pore-spanning membranes.

    PubMed

    Schwenen, Lando L G; Hubrich, Raphael; Milovanovic, Dragomir; Geil, Burkhard; Yang, Jian; Kros, Alexander; Jahn, Reinhard; Steinem, Claudia

    2015-07-13

    Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic.

  17. Resolving single membrane fusion events on planar pore-spanning membranes

    PubMed Central

    Schwenen, Lando L. G.; Hubrich, Raphael; Milovanovic, Dragomir; Geil, Burkhard; Yang, Jian; Kros, Alexander; Jahn, Reinhard; Steinem, Claudia

    2015-01-01

    Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic. PMID:26165860

  18. Membrane-disrupting iridium(iii) oligocationic organometallopeptides.

    PubMed

    Salvadó, Iria; Gamba, Ilaria; Montenegro, Javier; Martínez-Costas, José; Brea, Jose Manuel; Loza, María Isabel; Vázquez López, Miguel; Vázquez, M Eugenio

    2016-09-21

    A series of oligoarginine peptide derivatives containing cyclometallated iridium(iii) units display remarkable cytotoxicity, comparable to that of cisplatin. In vitro studies with unilamellar vesicles support a membrane-disrupting mechanism of action.

  19. Drug Delivery via Cell Membrane Fusion Using Lipopeptide Modified Liposomes

    PubMed Central

    2016-01-01

    Efficient delivery of drugs to living cells is still a major challenge. Currently, most methods rely on the endocytotic pathway resulting in low delivery efficiency due to limited endosomal escape and/or degradation in lysosomes. Here, we report a new method for direct drug delivery into the cytosol of live cells in vitro and invivo utilizing targeted membrane fusion between liposomes and live cells. A pair of complementary coiled-coil lipopeptides was embedded in the lipid bilayer of liposomes and cell membranes respectively, resulting in targeted membrane fusion with concomitant release of liposome encapsulated cargo including fluorescent dyes and the cytotoxic drug doxorubicin. Using a wide spectrum of endocytosis inhibitors and endosome trackers, we demonstrate that the major site of cargo release is at the plasma membrane. This method thus allows for the quick and efficient delivery of drugs and is expected to have many invitro, ex vivo, and invivo applications. PMID:27725960

  20. Simulation of polyethylene glycol and calcium-mediated membrane fusion

    SciTech Connect

    Pannuzzo, Martina; De Jong, Djurre H.; Marrink, Siewert J.; Raudino, Antonio

    2014-03-28

    We report on the mechanism of membrane fusion mediated by polyethylene glycol (PEG) and Ca{sup 2+} by means of a coarse-grained molecular dynamics simulation approach. Our data provide a detailed view on the role of cations and polymer in modulating the interaction between negatively charged apposed membranes. The PEG chains cause a reduction of the inter-lamellar distance and cause an increase in concentration of divalent cations. When thermally driven fluctuations bring the membranes at close contact, a switch from cis to trans Ca{sup 2+}-lipid complexes stabilizes a focal contact acting as a nucleation site for further expansion of the adhesion region. Flipping of lipid tails induces subsequent stalk formation. Together, our results provide a molecular explanation for the synergistic effect of Ca{sup 2+} and PEG on membrane fusion.

  1. Membrane fusion of Semliki Forest virus involves homotrimers of the fusion protein.

    PubMed Central

    Wahlberg, J M; Bron, R; Wilschut, J; Garoff, H

    1992-01-01

    Infection of cells with enveloped viruses is accomplished through membrane fusion. The binding and fusion processes are mediated by the spike proteins in the envelope of the virus particle and usually involve a series of conformational changes in these proteins. We have studied the low-pH-mediated fusion process of the alphavirus Semliki Forest virus (SFV). The spike protein of SFV is composed of three copies of the protein heterodimer E2E1. This structure is resistant to solubilization in mild detergents such as Nonidet P-40 (NP40). We have recently shown that the spike structure is reorganized during virus entry into acidic endosomes (J. M. Wahlberg and H. Garoff, J. Cell Biol. 116:339-348, 1992). The original NP40-resistant heterodimer is dissociated, and the E1 subunits form new NP40-resistant protein oligomers. Here, we show that the new oligomer is represented by an E1 trimer. From studies that use an in vitro assay for fusion of SFV with liposomes, we show that the E1 trimer is efficiently expressed during virus-mediated membrane fusion. Time course studies show that both E1 trimer formation and fusion are fast processes, occurring in seconds. It was also possible to inhibit virus binding and fusion with a monoclonal antibody directed toward the trimeric E1. These results give support for a model in which the E1 trimeric structure is involved in the SFV-mediated fusion reaction. Images PMID:1433520

  2. The Flocculating Cationic Polypetide from Moringa oleifera Seeds Damages Bacterial Cell Membranes by Causing Membrane Fusion.

    PubMed

    Shebek, Kevin; Schantz, Allen B; Sines, Ian; Lauser, Kathleen; Velegol, Stephanie; Kumar, Manish

    2015-04-21

    A cationic protein isolated from the seeds of the Moringa oleifera tree has been extensively studied for use in water treatment in developing countries and has been proposed for use in antimicrobial and therapeutic applications. However, the molecular basis for the antimicrobial action of this peptide, Moringa oleifera cationic protein (MOCP), has not been previously elucidated. We demonstrate here that a dominant mechanism of MOCP antimicrobial activity is membrane fusion. We used a combination of cryogenic electron microscopy (cryo-EM) and fluorescence assays to observe and study the kinetics of fusion of membranes in liposomes representing model microbial cells. We also conducted cryo-EM experiments on E. coli cells where MOCP was seen to fuse the inner and outer membranes. Coarse-grained molecular dynamics simulations of membrane vesicles with MOCP molecules were used to elucidate steps in peptide adsorption, stalk formation, and fusion between membranes.

  3. Distinct functional determinants of influenza hemagglutinin-mediated membrane fusion

    PubMed Central

    Ivanovic, Tijana; Harrison, Stephen C

    2015-01-01

    Membrane fusion is the critical step for infectious cell penetration by enveloped viruses. We have previously used single-virion measurements of fusion kinetics to study the molecular mechanism of influenza-virus envelope fusion. Published data on fusion inhibition by antibodies to the 'stem' of influenza virus hemagglutinin (HA) now allow us to incorporate into simulations the provision that some HAs are inactive. We find that more than half of the HAs are unproductive even for virions with no bound antibodies, but that the overall mechanism is extremely robust. Determining the fraction of competent HAs allows us to determine their rates of target-membrane engagement. Comparison of simulations with data from H3N2 and H1N1 viruses reveals three independent functional variables of HA-mediated membrane fusion closely linked to neutralization susceptibility. Evidence for compensatory changes in the evolved mechanism sets the stage for studies aiming to define the molecular constraints on HA evolvability. DOI: http://dx.doi.org/10.7554/eLife.11009.001 PMID:26613408

  4. Fusion of Sendai virus with vesicles of oligomerizable lipids: a microcalorimetric analysis of membrane fusion.

    PubMed

    Ravoo, B J; Weringa, W D; Engberts, J B

    2000-01-01

    Sendai virus fuses efficiently with small and large unilamellar vesicles of the lipid 1,2-di-n-hexadecyloxypropyl-4- (beta-nitrostyryl) phosphate (DHPBNS) at pH 7.4 and 37 degrees C, as shown by lipid mixing assays and electron microscopy. However, fusion is strongly inhibited by oligomerization of the head groups of DHPBNS in the bilayer vesicles. The enthalpy associated with fusion of Sendai virus with DHPBNS vesicles was measured by isothermal titration microcalorimetry, comparing titrations of Sendai virus into (i) solutions of DHPBNS vesicles (which fuse with the virus) and (ii) oligomerized DHPBNS vesicles (which do not fuse with the virus), respectively. The observed heat effect of fusion of Sendai virus with DHPBNS vesicles is strongly dependent on the buffer medium, reflecting a partial charge neutralization of the Sendai F and HN proteins upon insertion into the negatively-charged vesicle membrane. No buffer effect was observed for the titration of Sendai virus into oligomerized DHPBNS vesicles, indicating that inhibition of fusion is a result of inhibition of insertion of the fusion protein into the target membrane. Fusion of Sendai virus with DHPBNS vesicles is endothermic and entropy-driven. The positive enthalpy term is dominated by heat effects resulting from merging of the protein-rich viral envelope with the lipid vesicle bilayers rather than by the fusion of the viral with the vesicle bilayers per se.

  5. Polysulfone membranes. III. Performance evaluation of polyethersulfone-PVP membranes

    SciTech Connect

    Tam, C.M.; Matsuura, T.; Tweddle, T.A. ); Hazlett, J.D. )

    1993-12-01

    The performance of membranes produced from casting solutions consisting of polyethersulfone (PES), poly-(N-vinyl-pyrrolidone) (PVP), and N-methyl-2-pyrrolidinone (NMP) were systematically studied. Zero-shear casting solution viscosities for these polymer solutions were determined as a function of PES and PVP concentrations. Ultrafiltration membranes were then cast using the phase inversion technique and characterized by separation experiments using polyethylene glycols of various molecular weights as test solutes. A pore flow model was fitted to the resulting separation data to provide estimates of the average pore radius and membrane porosity. These parameters were used to compare laboratory results for this membrane casting solution system with performance data for commercially available polyethersulfone membranes. 15 refs., 4 figs., 1 tab.

  6. Membrane-spanning lipids for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer

    PubMed Central

    Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

    2015-01-01

    A Förster resonance energy transfer-based fusion and transfer assay was developed to study, in model membranes, protein-mediated membrane fusion and intermembrane lipid transfer of fluorescent sphingolipid analogs. For this assay, it became necessary to apply labeled reporter molecules that are resistant to spontaneous as well as protein-mediated intermembrane transfer. The novelty of this assay is the use of nonextractable fluorescent membrane-spanning bipolar lipids. Starting from the tetraether lipid caldarchaeol, we synthesized fluorescent analogs with fluorophores at both polar ends. In addition, we synthesized radioactive glycosylated caldarchaeols. These labeled lipids were shown to stretch through bilayer membranes rather than to loop within a single lipid layer of liposomes. More important, the membrane-spanning lipids (MSLs) in contrast to phosphoglycerides proved to be nonextractable by proteins. We could show that the GM2 activator protein (GM2AP) is promiscuous with respect to glycero- and sphingolipid transfer. Saposin (Sap) B also transferred sphingolipids albeit with kinetics different from GM2AP. In addition, we could unambiguously show that the recombinant activator protein Sap C x His6 induced membrane fusion rather than intermembrane lipid transfer. These findings showed that these novel MSLs, in contrast with fluorescent phosphoglycerolipids, are well suited for an uncompromised monitoring of membrane fusion and intermembrane lipid transfer. PMID:26269359

  7. Plant cytokinesis: a tale of membrane traffic and fusion.

    PubMed

    Jürgens, Gerd; Park, Misoon; Richter, Sandra; Touihri, Sonja; Krause, Cornelia; El Kasmi, Farid; Mayer, Ulrike

    2015-02-01

    Cytokinesis separates the forming daughter cells. Higher plants have lost the ability to constrict the plasma membrane (PM) in the division plane. Instead, trans-Golgi network (TGN)-derived membrane vesicles are targeted to the centre of the division plane and generate, by homotypic fusion, the partitioning membrane named cell plate (CP). The CP expands in a centrifugal fashion until its margin fuses with the PM at the cortical division site. Mutant screens in Arabidopsis have identified a cytokinesis-specific syntaxin named KNOLLE and an interacting Sec1/Munc18 (SM) protein named KEULE both of which are required for vesicle fusion during cytokinesis. KNOLLE is only made during M-phase, targeted to the division plane and degraded in the vacuole at the end of cytokinesis. Here we address mechanisms of KNOLLE trafficking and interaction of KNOLLE with different soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) partners and with SM-protein KEULE, ensuring membrane fusion in cytokinesis.

  8. Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension

    PubMed Central

    Bretou, Marine; Jouannot, Ouardane; Fanget, Isabelle; Pierobon, Paolo; Larochette, Nathanaël; Gestraud, Pierre; Guillon, Marc; Emiliani, Valentina; Gasman, Stéphane; Desnos, Claire; Lennon-Duménil, Ana-Maria; Darchen, François

    2014-01-01

    Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force. PMID:25143404

  9. Mechanistic Insight into Bunyavirus-Induced Membrane Fusion from Structure-Function Analyses of the Hantavirus Envelope Glycoprotein Gc

    PubMed Central

    Stettner, Eva; Jeffers, Scott Allen; Pérez-Vargas, Jimena; Pehau-Arnaudet, Gerard; Tortorici, M. Alejandra; Jestin, Jean-Luc; England, Patrick; Tischler, Nicole D.; Rey, Félix A.

    2016-01-01

    Hantaviruses are zoonotic viruses transmitted to humans by persistently infected rodents, giving rise to serious outbreaks of hemorrhagic fever with renal syndrome (HFRS) or of hantavirus pulmonary syndrome (HPS), depending on the virus, which are associated with high case fatality rates. There is only limited knowledge about the organization of the viral particles and in particular, about the hantavirus membrane fusion glycoprotein Gc, the function of which is essential for virus entry. We describe here the X-ray structures of Gc from Hantaan virus, the type species hantavirus and responsible for HFRS, both in its neutral pH, monomeric pre-fusion conformation, and in its acidic pH, trimeric post-fusion form. The structures confirm the prediction that Gc is a class II fusion protein, containing the characteristic β-sheet rich domains termed I, II and III as initially identified in the fusion proteins of arboviruses such as alpha- and flaviviruses. The structures also show a number of features of Gc that are distinct from arbovirus class II proteins. In particular, hantavirus Gc inserts residues from three different loops into the target membrane to drive fusion, as confirmed functionally by structure-guided mutagenesis on the HPS-inducing Andes virus, instead of having a single “fusion loop”. We further show that the membrane interacting region of Gc becomes structured only at acidic pH via a set of polar and electrostatic interactions. Furthermore, the structure reveals that hantavirus Gc has an additional N-terminal “tail” that is crucial in stabilizing the post-fusion trimer, accompanying the swapping of domain III in the quaternary arrangement of the trimer as compared to the standard class II fusion proteins. The mechanistic understandings derived from these data are likely to provide a unique handle for devising treatments against these human pathogens. PMID:27783711

  10. Vesicle fusion to planar membranes is enhanced by cholesterol and low temperature.

    PubMed

    Lee, David E; Lew, Matthew G; Woodbury, Dixon J

    2013-01-01

    Lipid composition and properties play an important role in many cellular properties such as fusion of vesicles to cell membranes, an essential process for exocytosis. Using a model system composed of artificial vesicles (liposomes) and artificial membranes (planar lipid bilayers), we observed that fusion is significantly affected by the lipid phase of the planar membrane. To determine the effect of lipid phases on fusion rates, we utilized the nystatin/ergosterol fusion assay and stimulated fusion with an osmotic gradient. Phase of the planar membrane was altered by changing cholesterol or temperature while the vesicular lipids were held constant. Liquid disordered (L(d) or L(α)) planar membranes were formed from phosphatidylethanolamine and phosphatidylcholine with unsaturated acyl chains. Addition of cholesterol shifts these membranes to the liquid ordered (L(o)) phase and increases liposome fusion. Planar membranes in the L(α) phase were also made from dipalmitoylphoshatidylcholine (DPPC) above the transition temperature (T(m)) of 41.5 ° C. Decreasing the temperature below T(m) shifts these membranes into the ripple phase (P(β')) and also increases liposome fusion. The cholesterol and temperature data are consistent with the hypothesis that fusion is promoted in membranes that have greater exposure of their lipid tails or in membranes which can form leaflet domains with negative curvature. The data are not consistent with the hypothesis that lipid mismatch drives fusion.

  11. An Ion Switch Regulates Fusion of Charged Membranes

    PubMed Central

    Siepi, Evgenios; Lutz, Silke; Meyer, Sylke; Panzner, Steffen

    2011-01-01

    Here we identify the recruitment of solvent ions to lipid membranes as the dominant regulator of lipid phase behavior. Our data demonstrate that binding of counterions to charged lipids promotes the formation of lamellar membranes, whereas their absence can induce fusion. The mechanism applies to anionic and cationic liposomes, as well as the recently introduced amphoteric liposomes. In the latter, an additional pH-dependent lipid salt formation between anionic and cationic lipids must occur, as indicated by the depletion of membrane-bound ions in a zone around pH 5. Amphoteric liposomes fuse under these conditions but form lamellar structures at both lower and higher pH values. The integration of these observations into the classic lipid shape theory yielded a quantitative link between lipid and solvent composition and the physical state of the lipid assembly. The key parameter of the new model, κ(pH), describes the membrane phase behavior of charged membranes in response to their ion loading in a quantitative way. PMID:21575575

  12. The Pre-Transmembrane Domain of the Autographa californica Multicapsid Nucleopolyhedrovirus GP64 Protein Is Critical for Membrane Fusion and Virus Infectivity▿ †

    PubMed Central

    Li, Zhaofei; Blissard, Gary W.

    2009-01-01

    The envelope glycoprotein, GP64, of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a class III viral fusion protein that mediates pH-triggered membrane fusion during virus entry. Viral fusion glycoproteins from many viruses contain a short region in the ectodomain and near the transmembrane domain, referred to as the pre-transmembrane (PTM) domain. In some cases, the PTM domain is rich in aromatic amino acids and plays an important role in membrane fusion. Although the 23-amino-acid (aa) PTM domain of AcMNPV GP64 lacks aromatic amino acids, we asked whether this region might also play a significant role in membrane fusion. We generated alanine scanning and single and multiple amino acid substitutions in the GP64 PTM domain. We specifically focused on amino acid positions conserved between baculovirus GP64 and thogotovirus GP75 proteins, as well as hydrophobic and charged amino acids. For each PTM-modified construct, we examined trimerization, cell surface localization, and membrane fusion activity. Membrane merger and pore formation were also examined. We identified eight aa positions that are important for membrane fusion activity. Critical positions were not clustered in the linear sequence but were distributed throughout the PTM domain. While charged residues were not critical or essential, three hydrophobic amino acids (L465, L476, and L480) played an important role in membrane fusion activity and appear to be involved in formation of the fusion pore. We also asked whether selected GP64 constructs were capable of rescuing a gp64null AcMNPV virus. These studies suggested that several conserved residues (T463, G460, G462, and G474) were not required for membrane fusion but were important for budding and viral infectivity. PMID:19692475

  13. Single residue deletions along the length of the influenza HA fusion peptide lead to inhibition of membrane fusion function

    SciTech Connect

    Langley, William A.; Thoennes, Sudha; Bradley, Konrad C.; Galloway, Summer E.; Talekar, Ganesh R.; Cummings, Sandra F.; Vareckova, Eva; Russell, Rupert J.; Steinhauer, David A.

    2009-11-25

    A panel of eight single amino acid deletion mutants was generated within the first 24 residues of the fusion peptide domain of the of the hemagglutinin (HA) of A/Aichi/2/68 influenza A virus (H3N2 subtype). The mutant HAs were analyzed for folding, cell surface transport, cleavage activation, capacity to undergo acid-induced conformational changes, and membrane fusion activity. We found that the mutant DELTAF24, at the C-terminal end of the fusion peptide, was expressed in a non-native conformation, whereas all other deletion mutants were transported to the cell surface and could be cleaved into HA1 and HA2 to activate membrane fusion potential. Furthermore, upon acidification these cleaved HAs were able to undergo the characteristic structural rearrangements that are required for fusion. Despite this, all mutants were inhibited for fusion activity based on two separate assays. The results indicate that the mutant fusion peptide domains associate with target membranes in a non-functional fashion, and suggest that structural features along the length of the fusion peptide are likely to be relevant for optimal membrane fusion activity.

  14. Influenza Virus-Mediated Membrane Fusion: Determinants of Hemagglutinin Fusogenic Activity and Experimental Approaches for Assessing Virus Fusion

    PubMed Central

    Hamilton, Brian S.; Whittaker, Gary R.; Daniel, Susan

    2012-01-01

    Hemagglutinin (HA) is the viral protein that facilitates the entry of influenza viruses into host cells. This protein controls two critical aspects of entry: virus binding and membrane fusion. In order for HA to carry out these functions, it must first undergo a priming step, proteolytic cleavage, which renders it fusion competent. Membrane fusion commences from inside the endosome after a drop in lumenal pH and an ensuing conformational change in HA that leads to the hemifusion of the outer membrane leaflets of the virus and endosome, the formation of a stalk between them, followed by pore formation. Thus, the fusion machinery is an excellent target for antiviral compounds, especially those that target the conserved stem region of the protein. However, traditional ensemble fusion assays provide a somewhat limited ability to directly quantify fusion partly due to the inherent averaging of individual fusion events resulting from experimental constraints. Inspired by the gains achieved by single molecule experiments and analysis of stochastic events, recently-developed individual virion imaging techniques and analysis of single fusion events has provided critical information about individual virion behavior, discriminated intermediate fusion steps within a single virion, and allowed the study of the overall population dynamics without the loss of discrete, individual information. In this article, we first start by reviewing the determinants of HA fusogenic activity and the viral entry process, highlight some open questions, and then describe the experimental approaches for assaying fusion that will be useful in developing the most effective therapies in the future. PMID:22852045

  15. The membrane-proximal region (MPR) of herpes simplex virus gB regulates association of the fusion loops with lipid membranes.

    PubMed

    Shelly, Spencer S; Cairns, Tina M; Whitbeck, J Charles; Lou, Huan; Krummenacher, Claude; Cohen, Gary H; Eisenberg, Roselyn J

    2012-11-20

    Glycoprotein B (gB), gD, and gH/gL constitute the fusion machinery of herpes simplex virus (HSV). Prior studies indicated that fusion occurs in a stepwise fashion whereby the gD/receptor complex activates the entire process, while gH/gL regulates the fusion reaction carried out by gB. Trimeric gB is a class III fusion protein. Its ectodomain of 773 amino acids contains a membrane-proximal region (MPR) (residues 731 to 773) and two fusion loops (FLs) per protomer. We hypothesized that the highly hydrophobic MPR interacts with the FLs, thereby masking them on virions until fusion begins. To test this hypothesis, we made a series of deletion, truncation, and point mutants of the gB MPR. Although the full-length deletion mutants were expressed in transfected cells, they were not transported to the cell surface, suggesting that removal of even small stretches of the MPR was highly detrimental to gB folding. To circumvent this limitation, we used a baculovirus expression system to generate four soluble proteins, each lacking the transmembrane region and cytoplasmic tail. All retained the FLs and decreasing portions of the MPR [gB(773t) (gB truncated at amino acid 773), gB(759t), gB(749t), and gB(739t)]. Despite the presence of the FLs, all were compromised in their ability to bind liposomes compared to the control, gB(730t), which lacks the MPR. We conclude that residues 731 to 739 are sufficient to mask the FLs, thereby preventing liposome association. Importantly, mutation of two aromatic residues (F732 and F738) to alanine restored the ability of gB(739t) to bind liposomes. Our data suggest that the MPR is important for modulating the association of gB FLs with target membranes. IMPORTANCE To successfully cause disease, a virus must infect host cells. Viral infection is a highly regulated, multistep process. For herpesviruses, genetic material transfers from the virus to the target cell through fusion of the viral and host cell lipid membranes. Here, we provide

  16. Tethering Membrane Fusion: Common and Different Players in Myoblasts and at the Synapse

    PubMed Central

    Rust, Marco B.; Jacob, Ralf; Renkawitz-Pohl, Renate

    2014-01-01

    Drosophila Membrane fusion is essential for the communication of membrane-defined compartments, development of multicellular organisms and tissue homeostasis. Although membrane fusion has been studied extensively, still little is known about the molecular mechanisms. Especially the intercellular fusion of cells during development and tissue homeostasis is poorly understood. Somatic muscle formation in Drosophila depends on the intercellular fusion of myoblasts. In this process, myoblasts recognize each other and adhere, thereby triggering a protein machinery that leads to electron-dense plaques, vesicles and F-actin formation at apposing membranes. Two models of how local membrane stress is achieved to induce the merging of the myoblast membranes have been proposed: the electron-dense vesicles transport and release a fusogen and F-actin bends the plasma membrane. In this review, we highlight cell-adhesion molecules and intracellular proteins known to be involved in myoblast fusion. The cell-adhesion proteins also mediate the recognition and adhesion of other cell types, such as neurons that communicate with each other via special intercellular junctions, termed chemical synapses. At these synapses, neurotransmitters are released through the intracellular fusion of synaptic vesicles with the plasma membrane. As the targeting of electron-dense vesicles in myoblasts shares some similarities with the targeting of synaptic vesicle fusion, we compare molecules required for synaptic vesicle fusion to recently identified molecules involved in myoblast fusion. PMID:24957080

  17. Analysis of residues near the fusion peptide in the influenza hemagglutinin structure for roles in triggering membrane fusion

    SciTech Connect

    Thoennes, Sudha; Li Zhunan; Lee, Byeong-Jae; Langley, William A.; Skehel, John J.; Russell, Rupert J.; Steinhauer, David A.

    2008-01-20

    Influenza virus entry occurs in endosomes, where acidification triggers irreversible conformational changes of the hemagglutinin glycoprotein (HA) that are required for membrane fusion. The acid-induced HA structural rearrangements have been well documented, and several models have been proposed to relate these to the process of membrane fusion. However, details regarding the role of specific residues in the initiation of structural rearrangements and membrane fusion are lacking. Here we report the results of studies on the HA of A/Aichi/2/68 virus (H3 subtype), in which mutants with changes at several ionizable residues in the vicinity of the 'fusion peptide' were analyzed for their effects on the pH at which conformational changes and membrane fusion occur. A variety of phenotypes was obtained, including examples of substitutions that lead to an increase in HA stability at reduced pH. Of particular note was the observation that a histidine to tyrosine substitution at HA1 position 17 resulted in a decrease in pH at which HA structural changes and membrane fusion take place by 0.3 relative to WT. The results are discussed in relation to possible mechanisms by which HA structural rearrangements are initiated at low pH and clade-specific differences near the fusion peptide.

  18. Revisit the Correlation between the Elastic Mechanics and Fusion of Lipid Membranes

    PubMed Central

    Fan, Zih-An; Tsang, Kuan-Yu; Chen, Si-Han; Chen, Yi-Fan

    2016-01-01

    Membrane fusion is a vital process in key cellular events. The fusion capability of a membrane depends on its elastic properties and varies with its lipid composition. It is believed that as the composition varies, the consequent change in C0 (monolayer spontaneous curvature) is the major factor dictating fusion, owing to the associated variation in GEs (elastic energies) of the fusion intermediates (e.g. stalk). By exploring the correlations among fusion, C0 and Kcp (monolayer bending modulus), we revisit this long-held belief and re-examine the fusogenic contributions of some relevant factors. We observe that not only C0 but also Kcp variations affect fusion, with depression in Kcp leading to suppression in fusion. Variations in GEs and inter-membrane interactions cannot account for the Kcp-fusion correlation; fusion is suppressed even as the GEs decrease with Kcp, indicating the presence of factor(s) with fusogenic importance overtaking that of GE. Furthermore, analyses find that the C0 influence on fusion is effected via modulating GE of the pre-fusion planar membrane, rather than stalk. The results support a recent proposition calling for a paradigm shift from the conventional view of fusion and may reshape our understanding to the roles of fusogenic proteins in regulating cellular fusion machineries. PMID:27534263

  19. Revisit the Correlation between the Elastic Mechanics and Fusion of Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Fan, Zih-An; Tsang, Kuan-Yu; Chen, Si-Han; Chen, Yi-Fan

    2016-08-01

    Membrane fusion is a vital process in key cellular events. The fusion capability of a membrane depends on its elastic properties and varies with its lipid composition. It is believed that as the composition varies, the consequent change in C0 (monolayer spontaneous curvature) is the major factor dictating fusion, owing to the associated variation in GEs (elastic energies) of the fusion intermediates (e.g. stalk). By exploring the correlations among fusion, C0 and Kcp (monolayer bending modulus), we revisit this long-held belief and re-examine the fusogenic contributions of some relevant factors. We observe that not only C0 but also Kcp variations affect fusion, with depression in Kcp leading to suppression in fusion. Variations in GEs and inter-membrane interactions cannot account for the Kcp-fusion correlation; fusion is suppressed even as the GEs decrease with Kcp, indicating the presence of factor(s) with fusogenic importance overtaking that of GE. Furthermore, analyses find that the C0 influence on fusion is effected via modulating GE of the pre-fusion planar membrane, rather than stalk. The results support a recent proposition calling for a paradigm shift from the conventional view of fusion and may reshape our understanding to the roles of fusogenic proteins in regulating cellular fusion machineries.

  20. Viral fusion protein transmembrane domain adopts β-strand structure to facilitate membrane topological changes for virus-cell fusion.

    PubMed

    Yao, Hongwei; Lee, Michelle W; Waring, Alan J; Wong, Gerard C L; Hong, Mei

    2015-09-01

    The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magic-angle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. (31)P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand-rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. (1)H-(31)P 2D correlation spectra and (2)H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation.

  1. Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures

    SciTech Connect

    Lamb, Robert A. . E-mail: ralamb@northwestern.edu; Paterson, Reay G.; Jardetzky, Theodore S.

    2006-01-05

    Paramyxoviruses enter cells by fusion of their lipid envelope with the target cell plasma membrane. Fusion of the viral membrane with the plasma membrane allows entry of the viral genome into the cytoplasm. For paramyxoviruses, membrane fusion occurs at neutral pH, but the trigger mechanism that controls the viral entry machinery such that it occurs at the right time and in the right place remains to be elucidated. Two viral glycoproteins are key to the infection process-an attachment protein that varies among different paramyxoviruses and the fusion (F) protein, which is found in all paramyxoviruses. For many of the paramyxoviruses (parainfluenza viruses 1-5, mumps virus, Newcastle disease virus and others), the attachment protein is the hemagglutinin/neuraminidase (HN) protein. In the last 5 years, atomic structures of paramyxovirus F and HN proteins have been reported. The knowledge gained from these structures towards understanding the mechanism of viral membrane fusion is described.

  2. Interactions between genes involved in exocytotic membrane fusion in paramecium.

    PubMed

    Bonnemain, H; Gulik-Krzywicki, T; Grandchamp, C; Cohen, J

    1992-03-01

    Crosses between members of two independent collections of Paramecium tetraurelia mutants blocked in the final membrane fusion step of trichocyst release (nd mutants) allowed us to define 13 complementation groups comprising 23 alleles. The mutant nd9a was then used as a target in a mutagenesis experiment designed to screen both revertants and new mutants in order to identify interacting genes. This mutant was chosen because it is the best known of its class to date and seems to be altered in assembly of the material connecting the trichocyst membrane to the plasma membrane and in assembly of the "rosette," a complex array of intramembranous particles in the plasma membrane at the trichocyst insertion sites. No revertants were obtained but two new mutants deficient for rosette assembly were identified, nd16b and nd18, whose gene products appear to interact with that of nd9. Indeed, the double mutants grown at 18 degrees, a permissive temperature for each of the single mutants, are characterized by a deficiency in exocytosis and in rosette assembly, as are also double mutants combining other allelic forms of the same genes. Moreover, aberrant dominance relationships among alleles of nd9 and of nd16 indicate the existence of interactions between identical subunits, which most likely assemble into multimeric structures. The nd16 gene product was shown by microinjection experiments to be a cytosolic factor, as is the nd9 gene product. It is therefore tempting to propose that the nd16 gene product also belongs to the connecting material and is involved in rosette assembly, in cooperation with nd9 and nd18.

  3. Acidification triggers Andes hantavirus membrane fusion and rearrangement of Gc into a stable post-fusion homotrimer.

    PubMed

    Acuña, Rodrigo; Bignon, Eduardo A; Mancini, Roberta; Lozach, Pierre-Yves; Tischler, Nicole D

    2015-11-01

    The hantavirus membrane fusion process is mediated by the Gc envelope glycoprotein from within endosomes. However, little is known about the specific mechanism that triggers Gc fusion activation, and its pre- and post-fusion conformations. We established cell-free in vitro systems to characterize hantavirus fusion activation. Low pH was sufficient to trigger the interaction of virus-like particles with liposomes. This interaction was dependent on a pre-fusion glycoprotein arrangement. Further, low pH induced Gc multimerization changes leading to non-reversible Gc homotrimers. These trimers were resistant to detergent, heat and protease digestion, suggesting characteristics of a stable post-fusion structure. No acid-dependent oligomerization rearrangement was detected for the trypsin-sensitive Gn envelope glycoprotein. Finally, acidification induced fusion of glycoprotein-expressing effector cells with non-susceptible CHO cells. Together, the data provide novel information on the Gc fusion trigger and its non-reversible activation involving lipid interaction, multimerization changes and membrane fusion which ultimately allow hantavirus entry into cells.

  4. Influenza viral membrane fusion is sensitive to sterol concentration but surprisingly robust to sterol chemical identity

    PubMed Central

    Zawada, Katarzyna E.; Wrona, Dominik; Rawle, Robert J.; Kasson, Peter M.

    2016-01-01

    Influenza virions are enriched in cholesterol relative to the plasma membrane from which they bud. Previous work has shown that fusion between influenza virus and synthetic liposomes is sensitive to the amount of cholesterol in either the virus or the target membrane. Here, we test the chemical properties of cholesterol required to promote influenza fusion by replacing cholesterol with other sterols and assaying viral fusion kinetics. We find that influenza fusion with liposomes is surprisingly robust to sterol chemical identity, showing no significant dependence on sterol identity in target membranes for any of the sterols tested. In the viral membrane, lanosterol slowed fusion somewhat, while polar sterols produced a more pronounced slowing and inhibition of fusion. No other sterols tested showed a significant perturbation in fusion rates, including ones previously shown to alter membrane bending moduli or phase behavior. Although fusion rates depend on viral cholesterol, they thus do not require cholesterol’s ability to support liquid-liquid phase coexistence. Using electron cryo-microscopy, we further find that sterol-dependent changes to hemagglutinin spatial patterning in the viral membrane do not require liquid-liquid phase coexistence. We therefore speculate that local sterol-hemagglutinin interactions in the viral envelope may control the rate-limiting step of fusion. PMID:27431907

  5. Low-dose ionizing radiation induces mitochondrial fusion and increases expression of mitochondrial complexes I and III in hippocampal neurons

    PubMed Central

    Chang, Chuang-Rung; Kao, Mou-Chieh; Chen, Kuan-Wei; Chiu, Shih-Che; Hsu, Ming-Ling; Hsiang, I-Chou; Chen, Yu-Jen; Chen, Linyi

    2015-01-01

    High energy ionizing radiation can cause DNA damage and cell death. During clinical radiation therapy, the radiation dose could range from 15 to 60 Gy depending on targets. While 2 Gy radiation has been shown to cause cancer cell death, studies also suggest a protective potential by low dose radiation. In this study, we examined the effect of 0.2-2 Gy radiation on hippocampal neurons. Low dose 0.2 Gy radiation treatment increased the levels of MTT. Since hippocampal neurons are post-mitotic, this result reveals a possibility that 0.2 Gy irradiation may increase mitochondrial activity to cope with stimuli. Maintaining neural plasticity is an energy-demanding process that requires high efficient mitochondrial function. We thus hypothesized that low dose radiation may regulate mitochondrial dynamics and function to ensure survival of neurons. Our results showed that five days after 0.2 Gy irradiation, no obvious changes on neuronal survival, neuronal synapses, membrane potential of mitochondria, reactive oxygen species levels, and mitochondrial DNA copy numbers. Interestingly, 0.2 Gy irradiation promoted the mitochondria fusion, resulting in part from the increased level of a mitochondrial fusion protein, Mfn2, and inhibition of Drp1 fission protein trafficking to the mitochondria. Accompanying with the increased mitochondrial fusion, the expressions of complexes I and III of the electron transport chain were also increased. These findings suggest that, hippocampal neurons undergo increased mitochondrial fusion to modulate cellular activity as an adaptive mechanism in response to low dose radiation. PMID:26415228

  6. Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

    SciTech Connect

    Kelsey, D.R.; Flanagan, T.D.; Young, J.E.; Yeagle, P.L. )

    1991-06-01

    Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18). This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. {sup 31}P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not.

  7. Selective transport of Fe(III) using ionic imprinted polymer (IIP) membrane particle

    NASA Astrophysics Data System (ADS)

    Djunaidi, Muhammad Cholid; Jumina, Siswanta, Dwi; Ulbricht, Mathias

    2015-12-01

    The membrane particles was prepared from polyvinyl alcohol (PVA) and polymer IIP with weight ratios of 1: 2 and 1: 1 using different adsorbent templates and casting thickness. The permeability of membrane towards Fe(III) and also mecanism of transport were studied. The selectivity of the membrane for Fe(III) was studied by performing adsorption experiments also with Cr(III) separately. In this study, the preparation of Ionic Imprinted Polymer (IIP) membrane particles for selective transport of Fe (III) had been done using polyeugenol as functional polymer. Polyeugenol was then imprinted with Fe (III) and then crosslinked with PEGDE under alkaline condition to produce polyeugenol-Fe-PEGDE polymer aggregates. The agrregates was then crushed and sieved using mesh size of 80 and the powder was then used to prepare the membrane particles by mixing it with PVA (Mr 125,000) solution in 1-Methyl-2-pyrrolidone (NMP) solvent. The membrane was obtained after casting at a speed of 25 m/s and soaking in NaOH solution overnight. The membrane sheet was then cut and Fe(III) was removed by acid to produce IIP membrane particles. Analysis of the membrane and its constituent was done by XRD, SEM and size selectivity test. Experimental results showed the transport of Fe(III) was faster with the decrease of membrane thickness, while the higher concentration of template ion correlates with higher Fe(III) being transported. However, the transport of Fe(III) was slower for higher concentration of PVA in the membrane. IImparticles works through retarded permeation mechanism, where Fe(III) was bind to the active side of IIP. The active side of IIP membrane was dominated by the -OH groups. The selectivity of all IIP membranes was confirmed as they were all unable to transport Cr (III), while NIP (Non-imprinted Polymer) membrane was able transport Cr (III).

  8. Fusion of phospholipid vesicles arrested by quick-freezing. The question of lipidic particles as intermediates in membrane fusion.

    PubMed

    Bearer, E L; Düzgünes, N; Friend, D S; Papahadjopoulos, D

    1982-12-01

    We have examined the early events in Ca2+-induced fusion of large (0.2 microns diameter) unilamellar cardiolipin/phosphatidylcholine and phosphatidylserine/phosphatidylethanolamine vesicles by quick-freezing freeze-fracture electron microscopy, eliminating the necessity of using glycerol as a cryoprotectant. Freeze-fracture replicas of vesicle suspensions frozen after 1-2 s of stimulation revealed that the majority of vesicles had already undergone membrane fusion, as evidenced by dumbbell-shaped structures and large vesicles. In the absence of glycerol, lipidic particles or the hexagonal HII phase, which have been proposed to be intermediate structures in membrane fusion, were not observed at the sites of fusion. Lipidic particles were evident in less than 5% of the cardiolipin/phosphatidylcholine vesicles after long-term incubation with Ca2+, and the addition of glycerol produced more vesicles displaying the particles. We have also shown that rapid fusion occurred within seconds of Ca2+ addition by the time-course of fluorescence emission produced by the intermixing of aqueous contents of two separate vesicle populations. These studies therefore have produced no evidence that lipidic particles are necessary intermediates for membrane fusion. On the contrary, they indicate that lipidic particles are structures obtained at equilibrium long after fusion has occurred and they become particularly prevalent in the presence of glycerol.

  9. Fusion protein of retinol-binding protein and albumin domain III reduces liver fibrosis.

    PubMed

    Lee, Hongsik; Jeong, Hyeyeun; Park, Sangeun; Yoo, Wonbaek; Choi, Soyoung; Choi, Kyungmin; Lee, Min-Goo; Lee, Mihwa; Cha, DaeRyong; Kim, Young-Sik; Han, Jeeyoung; Kim, Wonkon; Park, Sun-Hwa; Oh, Junseo

    2015-04-11

    Activated hepatic stellate cells (HSCs) play a key role in liver fibrosis, and inactivating HSCs has been considered a promising therapeutic approach. We previously showed that albumin and its derivative designed for stellate cell-targeting, retinol-binding protein-albumin domain III fusion protein (referred to as R-III), inactivate cultured HSCs. Here, we investigated the mechanism of action of albumin/R-III in HSCs and examined the anti-fibrotic potential of R-III in vivo. R-III treatment and albumin expression downregulated retinoic acid (RA) signaling which was involved in HSC activation. RA receptor agonist and retinaldehyde dehydrogenase overexpression abolished the anti-fibrotic effect of R-III and albumin, respectively. R-III uptake into cultured HSCs was significantly decreased by siRNA-STRA6, and injected R-III was localized predominantly in HSCs in liver. Importantly, R-III administration reduced CCl4- and bile duct ligation-induced liver fibrosis. R-III also exhibited a preventive effect against CCl4-inducd liver fibrosis. These findings suggest that the anti-fibrotic effect of albumin/R-III is, at least in part, mediated by downregulation of RA signaling and that R-III is a good candidate as a novel anti-fibrotic drug.

  10. The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction.

    PubMed

    Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J

    2015-03-01

    Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction.

  11. The cytoplasmic domain of the gamete membrane fusion protein HAP2 targets the protein to the fusion site in Chlamydomonas and regulates the fusion reaction

    PubMed Central

    Liu, Yanjie; Pei, Jimin; Grishin, Nick; Snell, William J.

    2015-01-01

    Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. PMID:25655701

  12. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets

    PubMed Central

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated. PMID:27517914

  13. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    PubMed

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated. PMID:27517914

  14. Importin beta negatively regulates nuclear membrane fusion and nuclear pore complex assembly.

    PubMed

    Harel, Amnon; Chan, Rene C; Lachish-Zalait, Aurelie; Zimmerman, Ella; Elbaum, Michael; Forbes, Douglass J

    2003-11-01

    Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin beta negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin beta is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin beta down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin beta to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin beta 45-462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin beta-regulation of NPC assembly. Excess full-length importin beta and beta 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin beta NPC block, which maps downstream of GTPgammaS and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 microM RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin beta. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.

  15. Fluorescence assays to monitor membrane fusion: potential application in biliary lipid secretion and vesicle interactions.

    PubMed

    Hoekstra, D

    1990-09-01

    Membrane fusion constitutes an essential, intermediate step in numerous cell biological processes, occurring for example during endocytosis, membrane recycling and exocytosis. Also less desirable events such as the infection of cells by animal viruses are mediated by membrane fusion during which the viral envelope merges with a cellular membrane, causing the expulsion of the viral nucleocapsid into the cytoplasm of the cell as an initial step in virus replication. Much of our current knowledge concerning the mechanism of membrane fusion has been derived from studies using simple artificial membranes, such as liposomes or phospholipid vesicles, as model systems. A most essential feature of these studies has been the development of membrane fusion assays that register in a sensitive and continuous fashion the mixing of membranes or the aqueous volumes initially enclosed by these membranes. Not only do these assays allow one to readily detect and quantify fusion, but they also provide the possibility to relate the kinetics of fusion to the rate by which certain molecular changes in membranes take place. Obviously, this insight is of relevance for understanding the mechanism of membrane fusion. The principles and applications of some representative assays that rely on the use of fluorescence spectroscopy will be discussed. Assays that monitor membrane mixing are commonly based on the detection of changes in resonance energy transfer efficiency or the relief of fluorescence self-quenching of appropriate fluorescent lipid analogs. Contents mixing assays rely on either the formation of a (aqueous-soluble) fluorescent complex or quenching of a fluorophore, encapsulated in one vesicle population, by a suitable quencher, entrapped in a second population.(ABSTRACT TRUNCATED AT 250 WORDS)

  16. FUSION-COMPETENT STATE INDUCED BY A C-TERMINAL HIV-1 FUSION PEPTIDE IN CHOLESTEROL-RICH MEMBRANES

    PubMed Central

    Apellániz, Beatriz; Nieva, José L.

    2015-01-01

    The replicative cycle of the Human Immunodeficiency Virus type-1 begins after fusion of the viral and target-cell membranes. The envelope glycoprotein gp41 transmembrane subunit contains conserved hydrophobic domains that engage and perturb the merging lipid bilayers. In this work, we have characterized the fusion-committed state generated in vesicles by CpreTM, a synthetic peptide derived from the sequence connecting the membrane-proximal external region (MPER) and the transmembrane domain (TMD) of gp41. Pre-loading cholesterol-rich vesicles with CpreTM rendered them competent for subsequent lipid-mixing with fluorescently-labeled target vesicles. Highlighting the physiological relevance of the lasting fusion-competent state, the broadly neutralizing antibody 4E10 bound to the CpreTM-primed vesicles and inhibited lipid-mixing. Heterotypic fusion assays disclosed dependence on the lipid composition of the vesicles that acted either as virus or cell membrane surrogates. Lipid-mixing exhibited above all a critical dependence on the cholesterol content in those experiments. We infer that the fusion-competent state described herein resembles bona-fide perturbations generated by the pre-hairpin MPER-TMD connection within the viral membrane. PMID:25617671

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

    PubMed Central

    Cox, Reagan G.; Mainou, Bernardo A.; Johnson, Monika; Hastings, Andrew K.; Schuster, Jennifer E.; Dermody, Terence S.; Williams, John V.

    2015-01-01

    Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments. PMID:26629703

  18. Characterization of electric field-induced fusion in erythrocyte ghost membranes

    PubMed Central

    1984-01-01

    Fusion has been reported to occur in a variety of membrane systems in response to the application of certain electric currents to the medium (Zimmermann, U., 1982, Biochim. Biophys. Acta., 694:227-277). The application of a weak but continuous alternating current causes the membranes in suspension to become rearranged into the "pearl-chain" formation. Fusion can then be induced by one or more strong direct current pulses that cause pore formation. This results in the conversion of individual membranes in the "pearl-chain" formation to a single membrane with one or more hourglass constrictions that form lumens which connect the cytoplasmic compartments. As the diameter of the lumens increases, the overall membrane shape grows to one large sphere. To further characterize electric field-induced fusion, experiments were conducted using the erythrocyte ghost as a model membrane, and a new combination of electrical circuit and fusion chamber that is simpler and improved over previous systems. All odd- shaped ghosts (collapsed or partly collapsed spherical shapes, echinocytes, discocytes, and stomatocytes) in 30 mM phosphate buffer was first converted to spherocytes and then fused with increasing yields by increasing the number of pulses. After fusion, the lateral diffusion of a fluorescent lipid soluble label (Dil) from labeled to unlabeled membranes was observed to occur both with and without the appearance in phase-contrast optics of distinct communication (lumens) between cytoplasmic compartments of the fused membranes. Connections between cytoplasmic compartments, however, were unmistakable with the instant transfer of a fluorescent water-soluble label (fluorescein isothiocyanate-dextran) from labeled to unlabeled cytoplasmic compartments upon fusion. Although pulses still resulted in the lateral diffusion of Dil to unlabeled membranes, the presence of glycerol in the medium strongly reduced the yield of lumens observable by phase- contrast optics in fusion events

  19. The Membrane-Proximal Region (MPR) of Herpes Simplex Virus gB Regulates Association of the Fusion Loops with Lipid Membranes

    PubMed Central

    Shelly, Spencer S.; Cairns, Tina M.; Whitbeck, J. Charles; Lou, Huan; Krummenacher, Claude; Cohen, Gary H.; Eisenberg, Roselyn J.

    2012-01-01

    ABSTRACT Glycoprotein B (gB), gD, and gH/gL constitute the fusion machinery of herpes simplex virus (HSV). Prior studies indicated that fusion occurs in a stepwise fashion whereby the gD/receptor complex activates the entire process, while gH/gL regulates the fusion reaction carried out by gB. Trimeric gB is a class III fusion protein. Its ectodomain of 773 amino acids contains a membrane-proximal region (MPR) (residues 731 to 773) and two fusion loops (FLs) per protomer. We hypothesized that the highly hydrophobic MPR interacts with the FLs, thereby masking them on virions until fusion begins. To test this hypothesis, we made a series of deletion, truncation, and point mutants of the gB MPR. Although the full-length deletion mutants were expressed in transfected cells, they were not transported to the cell surface, suggesting that removal of even small stretches of the MPR was highly detrimental to gB folding. To circumvent this limitation, we used a baculovirus expression system to generate four soluble proteins, each lacking the transmembrane region and cytoplasmic tail. All retained the FLs and decreasing portions of the MPR [gB(773t) (gB truncated at amino acid 773), gB(759t), gB(749t), and gB(739t)]. Despite the presence of the FLs, all were compromised in their ability to bind liposomes compared to the control, gB(730t), which lacks the MPR. We conclude that residues 731 to 739 are sufficient to mask the FLs, thereby preventing liposome association. Importantly, mutation of two aromatic residues (F732 and F738) to alanine restored the ability of gB(739t) to bind liposomes. Our data suggest that the MPR is important for modulating the association of gB FLs with target membranes. PMID:23170000

  20. Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

    PubMed

    Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

    2009-05-01

    Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

  1. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion

    PubMed Central

    Garcia, Natalie K.; Lee, Kelly K.

    2016-01-01

    The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system. PMID:26761026

  2. Dynamic Viral Glycoprotein Machines: Approaches for Probing Transient States That Drive Membrane Fusion.

    PubMed

    Garcia, Natalie K; Lee, Kelly K

    2016-01-11

    The fusion glycoproteins that decorate the surface of enveloped viruses undergo dramatic conformational changes in the course of engaging with target cells through receptor interactions and during cell entry. These refolding events ultimately drive the fusion of viral and cellular membranes leading to delivery of the genetic cargo. While well-established methods for structure determination such as X-ray crystallography have provided detailed structures of fusion proteins in the pre- and post-fusion fusion states, to understand mechanistically how these fusion glycoproteins perform their structural calisthenics and drive membrane fusion requires new analytical approaches that enable dynamic intermediate states to be probed. Methods including structural mass spectrometry, small-angle X-ray scattering, and electron microscopy have begun to provide new insight into pathways of conformational change and fusion protein function. In combination, the approaches provide a significantly richer portrait of viral fusion glycoprotein structural variation and fusion activation as well as inhibition by neutralizing agents. Here recent studies that highlight the utility of these complementary approaches will be reviewed with a focus on the well-characterized influenza virus hemagglutinin fusion glycoprotein system.

  3. Association of the fusion protein NSF with clathrin-coated vesicle membranes.

    PubMed Central

    Steel, G J; Tagaya, M; Woodman, P G

    1996-01-01

    N-ethylmaleimide-sensitive fusion protein (NSF) is a component of intracellular transport reactions. In order to understand the role of NSF during the fusion of endocytic transport vesicles with the endosome, we have investigated the binding of NSF to purified clathrin-coated vesicle components. First, we have examined whether detergent-solubilized coated vesicle membranes will support formation of NSF-containing 'fusion complexes'. Our results show that these membranes are substantially enriched in components capable of driving formation of these complexes, when compared with membranes from other sources. Secondly, we have analysed coated vesicle preparations for their NSF content. Coated vesicle preparations contain significant amounts of NSF. This was shown to be associated with coated vesicles rather than contaminating membranes by a number of criteria, and was found to be bound in an ATP-independent manner. These findings are discussed in the light of current models for vesicle fusion. Images PMID:8631296

  4. Characterization of HCoV-229E fusion core: Implications for structure basis of coronavirus membrane fusion

    SciTech Connect

    Liu Cheng; Feng Youjun; Gao Feng; Zhang Qiangmin; Wang Ming . E-mail: vetdean@cau.edu.cn

    2006-07-07

    Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimer of coiled-coil heterodimer (six {alpha}-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection.

  5. Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

    SciTech Connect

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao

    2013-04-05

    Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

  6. High Cholesterol Obviates a Prolonged Hemifusion Intermediate in Fast SNARE-Mediated Membrane Fusion

    PubMed Central

    Kreutzberger, Alex J.B.; Kiessling, Volker; Tamm, Lukas K.

    2015-01-01

    Cholesterol is essential for exocytosis in secretory cells, but the exact molecular mechanism by which it facilitates exocytosis is largely unknown. Distinguishing contributions from the lateral organization and dynamics of membrane proteins to vesicle docking and fusion and the promotion of fusion pores by negative intrinsic spontaneous curvature and other mechanical effects of cholesterol have been elusive. To shed more light on this process, we examined the effect of cholesterol on SNARE-mediated membrane fusion in a single-vesicle assay that is capable of resolving docking and elementary steps of fusion with millisecond time resolution. The effect of cholesterol on fusion pore formation between synaptobrevin-2 (VAMP-2)-containing proteoliposomes and acceptor t-SNARE complex-containing planar supported bilayers was examined using both membrane and content fluorescent markers. This approach revealed that increasing cholesterol in either the t-SNARE or the v-SNARE membrane favors a mechanism of direct fusion pore opening, whereas low cholesterol favors a mechanism leading to a long-lived (>5 s) hemifusion state. The amount of cholesterol in the target membrane had no significant effect on docking of synaptobrevin vesicles. Comparative studies with α-tocopherol (vitamin E) show that the negative intrinsic spontaneous curvature of cholesterol and its presumed promotion of a very short-lived (<50 ms) lipid stalk intermediate is the main factor that favors rapid fusion pore opening at high cholesterol. This study also shows that this single-vesicle fusion assay can distinguish between hemifusion and full fusion with only a single lipid dye, thereby freeing up a fluorescence channel for the simultaneous measurement of another parameter in fast time-resolved fusion assays. PMID:26200867

  7. Protein-lipid interactions and non-lamellar lipidic structures in membrane pore formation and membrane fusion.

    PubMed

    Gilbert, Robert J C

    2016-03-01

    Pore-forming proteins and peptides act on their targeted lipid bilayer membranes to increase permeability. This approach to the modulation of biological function is relevant to a great number of living processes, including; infection, parasitism, immunity, apoptosis, development and neurodegeneration. While some pore-forming proteins/peptides assemble into rings of subunits to generate discrete, well-defined pore-forming structures, an increasing number is recognised to form pores via mechanisms which co-opt membrane lipids themselves. Among these, membrane attack complex-perforin/cholesterol-dependent cytolysin (MACPF/CDC) family proteins, Bax/colicin family proteins and actinoporins are especially prominent and among the mechanisms believed to apply are the formation of non-lamellar (semi-toroidal or toroidal) lipidic structures. In this review I focus on the ways in which lipids contribute to pore formation and contrast this with the ways in which lipids are co-opted also in membrane fusion and fission events. A variety of mechanisms for pore formation that involve lipids exists, but they consistently result in stable hybrid proteolipidic structures. These structures are stabilised by mechanisms in which pore-forming proteins modify the innate capacity of lipid membranes to respond to their environment, changing shape and/or phase and binding individual lipid molecules directly. In contrast, and despite the diversity in fusion protein types, mechanisms for membrane fusion are rather similar to each other, mapping out a pathway from pairs of separated compartments to fully confluent fused membranes. Fusion proteins generate metastable structures along the way which, like long-lived proteolipidic pore-forming complexes, rely on the basic physical properties of lipid bilayers. Membrane fission involves similar intermediates, in the reverse order. I conclude by considering the possibility that at least some pore-forming and fusion proteins are evolutionarily related

  8. G-rich, a Drosophila selenoprotein, is a Golgi-resident type III membrane protein

    SciTech Connect

    Chen, Chang Lan; Shim, Myoung Sup; Chung, Jiyeol; Yoo, Hyun-Seung; Ha, Ji Min; Kim, Jin Young; Choi, Jinmi; Zang, Shu Liang; Hou, Xiao; Carlson, Bradley A.; Hatfield, Dolph L.; Lee, Byeong Jae . E-mail: imbglmg@plaza.snu.ac.kr

    2006-10-06

    G-rich is a Drosophila melanogaster selenoprotein, which is a homologue of human and mouse SelK. Subcellular localization analysis using GFP-tagged G-rich showed that G-rich was localized in the Golgi apparatus. The fusion protein was co-localized with the Golgi marker proteins but not with an endoplasmic reticulum (ER) marker protein in Drosophila SL2 cells. Bioinformatic analysis of G-rich suggests that this protein is either type II or type III transmembrane protein. To determine the type of transmembrane protein experimentally, GFP-G-rich in which GFP was tagged at the N-terminus of G-rich, or G-rich-GFP in which GFP was tagged at the C-terminus of G-rich, were expressed in SL2 cells. The tagged proteins were then digested with trypsin, and analyzed by Western blot analysis. The results showed that the C-terminus of the G-rich protein was exposed to the cytoplasm indicating it is a type III microsomal membrane protein. G-rich is First selenoprotein identified in the Golgi apparatus.

  9. Capturing Spontaneous Membrane Insertion of the Influenza Virus Hemagglutinin Fusion Peptide

    PubMed Central

    Baylon, Javier L.; Tajkhorshid, Emad

    2016-01-01

    Hemagglutinin (HA) is a protein located on the surface of the influenza virus that mediates viral fusion to the host cellular membrane. During the fusion process the HA fusion peptide (HAfp), formed by the first 23 N-terminal residues of HA and structurally characterized by two alpha helices (Helix A and Helix B) tightly packed in a hairpin-like arrangement, is the only part of the virus in direct contact with the host membrane. After encountering the host cell HAfp is believed to insert into the membrane, thereby initiating the fusion of the viral and host membranes. Detailed characterization of the interactions between the HAfp and cellular membrane is therefore of high relevance to the mechanism of viral entry into the host cell. Employing HMMM membrane representation with enhanced lipid mobility, we have performed a large set of independent simulations of unbiased membrane binding of HAfp. We have been able to capture spontaneous binding and insertion of HAfp consistently in nearly all the simulations. A reproducible membrane-bound configuration emerges from these simulations, despite employing a diverse set of initial configurations. Extension of several of the simulations into full membrane systems confirms the stability of the membrane-bound form obtained from HMMM binding simulations. The resulting model allows for the characterization of important interactions between the peptide and the membrane, and the details of the binding process of the peptide for the first time. Upon membrane binding, Helix A inserts much deeper into the membrane than Helix B, suggesting that the former is responsible for hydrophobic anchoring of the peptide into the membrane. Helix B, in contrast, is found to establish major amphipathic interactions at the interfacial region thereby contributing to binding strength of HAfp. PMID:25996559

  10. Regulation of mitochondrial inner membrane fusion: divergent evolution with similar solutions?

    PubMed

    Wagener, Johannes

    2016-05-01

    Continuous mitochondrial fusion and fission define the dynamic shape of mitochondria. One essential player of mitochondrial fusion is the conserved inner membrane dynamin-like GTPase Mgm1/OPA1. Limited proteolysis of this protein has been proposed as a mechanism to separate and subsequently eliminate dysfunctional parts from the mitochondrial network. Here, I briefly summarize our current knowledge about the underlying proteolytic processing steps in mammals, baker's yeast, Schizosaccharomyces pombe, Drosophila melanogaster and Aspergillus fumigatus. The apparent great diversity in Mgm1/OPA1 processing among the analyzed species indicates a surprising mechanistic heterogeneity in the regulation of mitochondrial inner membrane fusion. PMID:26613727

  11. Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

    PubMed

    Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

    2013-05-28

    Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

  12. Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

    SciTech Connect

    Pszon-Bartosz, Kamila; Hansen, Jesper S.; Stibius, Karin B.; Groth, Jesper S.; Helix-Nielsen, Claus

    2011-03-04

    Research highlights: {yields} We have established a vesicle fusion efficacy assay based on the major non-specific porin of Fusobacterium nucleatum (FomA). {yields} Maximal fusion obtained was almost 150,000 porin insertions during 20 min. {yields} Incorporation can be either first order or exponential kinetics which has implications for establishing protein delivery to biomimetic membranes. -- Abstract: Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR) = 50 more than 10{sup 5} FomA proteins could be incorporated in a bilayer array with a total membrane area of 2 mm{sup 2} within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.

  13. Interaction between La(III) and proteins on the plasma membrane of horseradish

    NASA Astrophysics Data System (ADS)

    Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

    2012-06-01

    Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

  14. A Substrate-Fusion Protein Is Trapped inside the Type III Secretion System Channel in Shigella flexneri

    PubMed Central

    Dohlich, Kim; Zumsteg, Anna Brotcke; Goosmann, Christian; Kolbe, Michael

    2014-01-01

    The Type III Secretion System (T3SS) is a macromolecular complex used by Gram-negative bacteria to secrete effector proteins from the cytoplasm across the bacterial envelope in a single step. For many pathogens, the T3SS is an essential virulence factor that enables the bacteria to interact with and manipulate their respective host. A characteristic structural feature of the T3SS is the needle complex (NC). The NC resembles a syringe with a basal body spanning both bacterial membranes and a long needle-like structure that protrudes from the bacterium. Based on the paradigm of a syringe-like mechanism, it is generally assumed that effectors and translocators are unfolded and secreted from the bacterial cytoplasm through the basal body and needle channel. Despite extensive research on T3SS, this hypothesis lacks experimental evidence and the mechanism of secretion is not fully understood. In order to elucidate details of the T3SS secretion mechanism, we generated fusion proteins consisting of a T3SS substrate and a bulky protein containing a knotted motif. Because the knot cannot be unfolded, these fusions are accepted as T3SS substrates but remain inside the NC channel and obstruct the T3SS. To our knowledge, this is the first time substrate fusions have been visualized together with isolated NCs and we demonstrate that substrate proteins are secreted directly through the channel with their N-terminus first. The channel physically encloses the fusion protein and shields it from a protease and chemical modifications. Our results corroborate an elementary understanding of how the T3SS works and provide a powerful tool for in situ-structural investigations in the future. This approach might also be applicable to other protein secretion systems that require unfolding of their substrates prior to secretion. PMID:24453973

  15. A capsid protein of nonenveloped Bluetongue virus exhibits membrane fusion activity.

    PubMed

    Forzan, Mario; Wirblich, Christoph; Roy, Polly

    2004-02-17

    The outer capsid layer of Bluetongue virus, a member of the nonenveloped Reoviridae family, is composed of two proteins, a receptor-binding protein, VP2, and a second protein, VP5, which shares structural features with class I fusion proteins of enveloped viruses. In the replication cycle of Bluetongue virus VP5 acts as a membrane permeabilization protein that mediates release of viral particles from endosomal compartments into the cytoplasm. Here, we show that VP5 can also act as a fusion protein and induce syncytium formation when it is fused to a transmembrane anchor and expressed on the cell surface. Fusion activity is strictly pH-dependent and is triggered by short exposure to low pH. No cell-cell fusion is observed at neutral pH. Deletion of the first 40 amino acids, which can fold into two amphipathic helices, abolishes fusion activity. Syncytium formation by VP5 is inhibited in the presence of VP2 when it is expressed in a membrane-anchored form. The data indicate an interaction between the outer capsid protein VP2 and VP5 and show that VP5 undergoes pH-dependent conformational changes that render it capable of interacting with cellular membranes. More importantly, our data show that a membrane permeabilization protein of a nonenveloped virus can evolve into a fusion protein by the addition of an appropriate transmembrane anchor. The results strongly suggest that the mechanism of membrane permeabilization by VP5 and membrane fusion by viral fusion proteins require similar structural features and conformational changes.

  16. Differential cargo mobilisation within Weibel-Palade bodies after transient fusion with the plasma membrane.

    PubMed

    Kiskin, Nikolai I; Babich, Victor; Knipe, Laura; Hannah, Matthew J; Carter, Tom

    2014-01-01

    Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼ 2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis. PMID:25233365

  17. Differential Cargo Mobilisation within Weibel-Palade Bodies after Transient Fusion with the Plasma Membrane

    PubMed Central

    Kiskin, Nikolai I.; Babich, Victor; Knipe, Laura; Hannah, Matthew J.; Carter, Tom

    2014-01-01

    Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis. PMID:25233365

  18. Cytosol-dependent membrane fusion in ER, nuclear envelope and nuclear pore assembly: biological implications.

    PubMed

    Rafikova, Elvira R; Melikov, Kamran; Chernomordik, Leonid V

    2010-01-01

    Endoplasmic reticulum and nuclear envelope rearrangements after mitosis are often studied in the reconstitution system based on Xenopus egg extract. In our recent work we partially replaced the membrane vesicles in the reconstitution mix with protein-free liposomes to explore the relative contributions of cytosolic and transmembrane proteins. Here we discuss our finding that cytosolic proteins mediate fusion between membranes lacking functional transmembrane proteins and the role of membrane fusion in endoplasmic reticulum and nuclear envelope reorganization. Cytosol-dependent liposome fusion has allowed us to restore, without adding transmembrane nucleoporins, functionality of nuclear pores, their spatial distribution and chromatin decondensation in nuclei formed at insufficient amounts of membrane material and characterized by only partial decondensation of chromatin and lack of nuclear transport. Both the mechanisms and the biological implications of the discovered coupling between spatial distribution of nuclear pores, chromatin decondensation and nuclear transport are discussed.

  19. Membrane topology of conserved components of the type III secretion system from the plant pathogen Xanthomonas campestris pv. vesicatoria.

    PubMed

    Berger, Carolin; Robin, Guillaume P; Bonas, Ulla; Koebnik, Ralf

    2010-07-01

    Type III secretion (T3S) systems play key roles in the assembly of flagella and the translocation of bacterial effector proteins into eukaryotic host cells. Eleven proteins which are conserved among gram-negative plant and animal pathogenic bacteria have been proposed to build up the basal structure of the T3S system, which spans both inner and outer bacterial membranes. We studied six conserved proteins, termed Hrc, predicted to reside in the inner membrane of the plant pathogen Xanthomonas campestris pv. vesicatoria. The membrane topology of HrcD, HrcR, HrcS, HrcT, HrcU and HrcV was studied by translational fusions to a dual alkaline phosphatase-beta-galactosidase reporter protein. Two proteins, HrcU and HrcV, were found to have the same membrane topology as the Yersinia homologues YscU and YscV. For HrcR, the membrane topology differed from the model for the homologue from Yersinia, YscR. For our data on three other protein families, exemplified by HrcD, HrcS and HrcT, we derived the first topology models. Our results provide what is believed to be the first complete model of the inner membrane topology of any bacterial T3S system and will aid in elucidating the architecture of T3S systems by ultrastructural analysis. PMID:20378646

  20. Bacteriophage membrane protein P9 as a fusion partner for the efficient expression of membrane proteins in Escherichia coli.

    PubMed

    Jung, Yuna; Jung, Hyeim; Lim, Dongbin

    2015-12-01

    Despite their important roles and economic values, studies of membrane proteins have been hampered by the difficulties associated with obtaining sufficient amounts of protein. Here, we report a novel membrane protein expression system that uses the major envelope protein (P9) of phage φ6 as an N-terminal fusion partner. Phage membrane protein P9 facilitated the synthesis of target proteins and their integration into the Escherichia coli cell membrane. This system was used to produce various multi-pass transmembrane proteins, including G-protein-coupled receptors, transporters, and ion channels of human origin. Green fluorescent protein fusion was used to confirm the correct folding of the expressed proteins. Of the 14 membrane proteins tested, eight were highly expressed, three were moderately expressed, and three were barely expressed in E. coli. Seven of the eight highly expressed proteins could be purified after extraction with the mild detergent lauryldimethylamine-oxide. Although a few proteins have previously been developed as fusion partners to augment membrane protein production, we believe that the major envelope protein P9 described here is better suited to the efficient expression of eukaryotic transmembrane proteins in E. coli.

  1. Disentangling Viral Membrane Fusion from Receptor Binding Using Synthetic DNA-Lipid Conjugates.

    PubMed

    Rawle, Robert J; Boxer, Steven G; Kasson, Peter M

    2016-07-12

    Enveloped viruses must bind to a receptor on the host membrane to initiate infection. Membrane fusion is subsequently initiated by a conformational change in the viral fusion protein, triggered by receptor binding, an environmental change, or both. Here, we present a strategy to disentangle the two processes of receptor binding and fusion using synthetic DNA-lipid conjugates to bind enveloped viruses to target membranes in the absence of receptor. This permits direct testing of whether receptor engagement affects the fusion mechanism as well as a comparison of fusion behavior across viruses with different receptor binding specificities. We demonstrate this approach by binding X-31 influenza virus to target vesicles and measuring the rates of individual pH-triggered lipid mixing events using fluorescence microscopy. Influenza lipid mixing kinetics are found to be independent of receptor binding, supporting the common yet previously unproven assumption that receptor binding does not produce any clustering or spatial rearrangement of viral hemagglutinin, which affects the rate-limiting step of pH-triggered fusion. This DNA-lipid tethering strategy should also allow the study of viruses where challenging receptor reconstitution has previously prevented single-virus fusion experiments. PMID:27410740

  2. Disentangling Viral Membrane Fusion from Receptor Binding Using Synthetic DNA-Lipid Conjugates.

    PubMed

    Rawle, Robert J; Boxer, Steven G; Kasson, Peter M

    2016-07-12

    Enveloped viruses must bind to a receptor on the host membrane to initiate infection. Membrane fusion is subsequently initiated by a conformational change in the viral fusion protein, triggered by receptor binding, an environmental change, or both. Here, we present a strategy to disentangle the two processes of receptor binding and fusion using synthetic DNA-lipid conjugates to bind enveloped viruses to target membranes in the absence of receptor. This permits direct testing of whether receptor engagement affects the fusion mechanism as well as a comparison of fusion behavior across viruses with different receptor binding specificities. We demonstrate this approach by binding X-31 influenza virus to target vesicles and measuring the rates of individual pH-triggered lipid mixing events using fluorescence microscopy. Influenza lipid mixing kinetics are found to be independent of receptor binding, supporting the common yet previously unproven assumption that receptor binding does not produce any clustering or spatial rearrangement of viral hemagglutinin, which affects the rate-limiting step of pH-triggered fusion. This DNA-lipid tethering strategy should also allow the study of viruses where challenging receptor reconstitution has previously prevented single-virus fusion experiments.

  3. The Influenza Hemagglutinin Fusion Domain Is an Amphipathic Helical Hairpin That Functions by Inducing Membrane Curvature*

    PubMed Central

    Smrt, Sean T.; Draney, Adrian W.; Lorieau, Justin L.

    2015-01-01

    The highly conserved N-terminal 23 residues of the hemagglutinin glycoprotein, known as the fusion peptide domain (HAfp23), is vital to the membrane fusion and infection mechanism of the influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We demonstrate that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk. PMID:25398882

  4. The influenza hemagglutinin fusion domain is an amphipathic helical hairpin that functions by inducing membrane curvature.

    PubMed

    Smrt, Sean T; Draney, Adrian W; Lorieau, Justin L

    2015-01-01

    The highly conserved N-terminal 23 residues of the hemagglutinin glycoprotein, known as the fusion peptide domain (HAfp23), is vital to the membrane fusion and infection mechanism of the influenza virus. HAfp23 has a helical hairpin structure consisting of two tightly packed amphiphilic helices that rest on the membrane surface. We demonstrate that HAfp23 is a new class of amphipathic helix that functions by leveraging the negative curvature induced by two tightly packed helices on membranes. The helical hairpin structure has an inverted wedge shape characteristic of negative curvature lipids, with a bulky hydrophobic region and a relatively small hydrophilic head region. The F3G mutation reduces this inverted wedge shape by reducing the volume of its hydrophobic base. We show that despite maintaining identical backbone structures and dynamics as the wild type HAfp23, the F3G mutant has an attenuated fusion activity that is correlated to its reduced ability to induce negative membrane curvature. The inverted wedge shape of HAfp23 is likely to play a crucial role in the initial stages of membrane fusion by stabilizing negative curvature in the fusion stalk.

  5. Reconstituting SNARE-mediated membrane fusion at the single liposome level.

    PubMed

    Kiessling, Volker; Liang, Binyong; Tamm, Lukas K

    2015-01-01

    Successful reconstitutions of SNARE-mediated intracellular membrane fusion have been achieved in bulk fusion assays since 1998 and in single liposome fusion assays since 2004. Especially in neuronal presynaptic SNARE-mediated exocytosis, fusion is controlled by numerous accessory proteins, of which some functions have also been reconstituted in vitro. The development of and results obtained with two fundamentally different single liposome fusion assays, namely liposome-to-supported membrane and liposome-to-liposome, are reviewed. Both assays distinguish between liposome docking and fusion steps of the overall fusion reaction and both assays are capable of resolving hemi-and full-fusion intermediates and end states. They have opened new windows for elucidating the mechanisms of these fundamentally important cellular reactions with unprecedented time and molecular resolution. Although many of the molecular actors in this process have been discovered, we have only scratched the surface of looking at their fascinating plays, interactions, and choreographies that lead to vesicle traffic as well as neurotransmitter and hormone release in the cell.

  6. Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

    PubMed

    Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J

    2010-12-01

    Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

  7. The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion.

    PubMed

    Lu, Bin

    2015-01-01

    Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

  8. The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion

    PubMed Central

    2015-01-01

    Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly. PMID:26157630

  9. Lysosome-membrane fusion mediated superoxide production in hyperglycaemia-induced endothelial dysfunction.

    PubMed

    Bao, Jun-Xiang; Chang, Hui; Lv, Yong-Gang; Yu, Jin-Wen; Bai, Yun-Gang; Liu, Huan; Cai, Yue; Wang, Ling; Ma, Jin; Chang, Yao-Ming

    2012-01-01

    Lysosomal exocytosis and fusion to cellular membrane is critical in the oxidative stress formation of endothelium under apoptotic stimulus. We investigated the role therein of it in hyperglycaemia-induced endothelial dysfunction. The lysosome-membrane fusion was shown by the expression of lamp1, the lysosomal membrane marker, on cellular membrane and the transportation of lysosomal symbolic enzymes into cultural medium. We also examined the ceramide production, lipid rafts (LRs) clustering, colocalization of gp91(phox), a NADPH oxidase subunit (NOX) to LRs clusters, superoxide (O₂·⁻) formation and nitric oxide (NO) content in human umbilical vein endothelial cells (HUVEC) and the endothelium-dependent NO-mediated vasodilation in isolated rat aorta. As compared to normal glucose (5.6 mmol/l, Ctrl) incubation, high glucose (22 mmol/l, HG) exposure facilitated the lysosome-membrane fusion in HUVEC shown by significantly increased quantity of lamp1 protein on cellular membrane and enhanced activity of lysosomal symbolized enzymes in cultural medium. HG incubation also elicited ceramide generation, LRs clustering and gp91(phox) colocalization to LRs clusters which were proved to mediate the HG induced O₂·⁻ formation and NO depletion in HUVEC. Functionally, the endothelium-dependent NO-mediated vasodilation in aorta was blunted substantially after HG incubation. Moreover, the HG-induced effect including ceramide production, LRs clustering, gp91(phox) colocalization to LRs clusters, O₂·⁻ formation and endothelial dysfunction could be blocked significantly by the inhibition of lysosome-membrane fusion. We propose that hyperglycaemia-induced endothelial impairment is closely related to the lysosome-membrane fusion and the following LRs clustering, LRs-NOX platforms formation and O₂·⁻ production. PMID:22253932

  10. NUCLEAR MEMBRANE FUSION IN FERTILIZED LYTECHINUS VARIEGATUS EGGS

    PubMed Central

    Aronson, John F.

    1973-01-01

    Fusion of apposed nuclear envelopes is frequently seen at telophase during postmitotic reorganization of the nucleus, but only rarely at other times in the cell cycle. We attempted to define an experimental system for studying changes in the nuclear envelope related to the cell cycle by varying the time of pronuclear apposition in fertilized Lytechinus variegatus eggs. This approach was based on the assumption that the period from fertilization to metaphase of the first cleavage division corresponds to the period from telophase to metaphase in the generalized cell cycle. The experimental approach used was to block the movement of the pronuclei with Colcemid and then to release this block at varying times after insemination by photochemically inactivating the Colcemid. The results show that apposed pronuclear envelopes can fuse from soon after insemination until the anticipated time of prometaphase. Fusion occurred in about 3 min as scored by light microscopy and this time did not vary significantly with the time after insemination. The potential for nuclear fusion is not restricted to pronuclei alone since diploid nuclei in binucleate cells could be fused using centrifugation in solutions of Colcemid to bring the nuclei into apposition. It is suggested that the potential for nuclear fusion is not necessarily related to the cell cycle and that modification of the nuclear envelope, possibly by association with chromatin or other fibrous material restricts nuclear fusion in most multinucleated cells. PMID:4125367

  11. Yeast lipin 1 orthologue pah1p regulates vacuole homeostasis and membrane fusion.

    PubMed

    Sasser, Terry; Qiu, Quan-Sheng; Karunakaran, Surya; Padolina, Mark; Reyes, Anna; Flood, Blake; Smith, Sheena; Gonzales, Chad; Fratti, Rutilio A

    2012-01-13

    Vacuole homotypic fusion requires a group of regulatory lipids that includes diacylglycerol, a fusogenic lipid that is produced through multiple metabolic pathways including the dephosphorylation of phosphatidic acid (PA). Here we examined the relationship between membrane fusion and PA phosphatase activity. Pah1p is the single yeast homologue of the Lipin family of PA phosphatases. Deletion of PAH1 was sufficient to cause marked vacuole fragmentation and abolish vacuole fusion. The function of Pah1p solely depended on its phosphatase activity as complementation studies showed that wild type Pah1p restored fusion, whereas the phosphatase dead mutant Pah1p(D398E) had no effect. We discovered that the lack of PA phosphatase activity blocked fusion by inhibiting the binding of SNAREs to Sec18p, an N-ethylmaleimide-sensitive factor homologue responsible for priming inactive cis-SNARE complexes. In addition, pah1Δ vacuoles were devoid of the late endosome/vacuolar Rab Ypt7p, the phosphatidylinositol 3-kinase Vps34p, and Vps39p, a subunit of the HOPS (homotypic fusion and vacuole protein sorting) tethering complex, all of which are required for vacuole fusion. The lack of Vps34p resulted in the absence of phosphatidylinositol 3-phosphate, a lipid required for SNARE activity and vacuole fusion. These findings demonstrate that Pah1p and PA phosphatase activity are critical for vacuole homeostasis and fusion.

  12. VAMP8-dependent fusion of recycling endosomes with the plasma membrane facilitates T lymphocyte cytotoxicity

    PubMed Central

    Marshall, Misty R.; Pattu, Varsha; Halimani, Mahantappa; Maier-Peuschel, Monika; Müller, Martha-Lena; Becherer, Ute; Hong, Wanjin; Hoth, Markus; Tschernig, Thomas

    2015-01-01

    Cytotoxic T lymphocytes (CTLs) eliminate infected and neoplastic cells through directed release of cytotoxic granule contents. Although multiple SNARE proteins have been implicated in cytotoxic granule exocytosis, the role of vesicular SNARE proteins, i.e., vesicle-associated membrane proteins (VAMPs), remains enigmatic. VAMP8 was posited to represent the cytotoxic granule vesicular SNARE protein mediating exocytosis in mice. In primary human CTLs, however, VAMP8 colocalized with Rab11a-positive recycling endosomes. Upon stimulation, these endosomes rapidly trafficked to and fused with the plasma membrane, preceding fusion of cytotoxic granules. Knockdown of VAMP8 blocked both recycling endosome and cytotoxic granule fusion at immune synapses, without affecting activating signaling. Mechanistically, VAMP8-dependent recycling endosomes deposited syntaxin-11 at immune synapses, facilitating assembly of plasma membrane SNARE complexes for cytotoxic granule fusion. Hence, cytotoxic granule exocytosis is a sequential, multivesicle fusion process requiring VAMP8-mediated recycling endosome fusion before cytotoxic granule fusion. Our findings imply that secretory granule exocytosis pathways in other cell types may also be more complex than previously appreciated. PMID:26124288

  13. Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins

    PubMed Central

    Brunger, Axel T.; Cipriano, Daniel J.; Diao, Jiajie

    2015-01-01

    Abstract Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed. PMID:25788028

  14. Homotypic vacuole fusion in yeast requires organelle acidification and not the V-ATPase membrane domain.

    PubMed

    Coonrod, Emily M; Graham, Laurie A; Carpp, Lindsay N; Carr, Tom M; Stirrat, Laura; Bowers, Katherine; Bryant, Nia J; Stevens, Tom H

    2013-11-25

    Studies of homotypic vacuole-vacuole fusion in the yeast Saccharomyces cerevisiae have been instrumental in determining the cellular machinery required for eukaryotic membrane fusion and have implicated the vacuolar H(+)-ATPase (V-ATPase). The V-ATPase is a multisubunit, rotary proton pump whose precise role in homotypic fusion is controversial. Models formulated from in vitro studies suggest that it is the proteolipid proton-translocating pore of the V-ATPase that functions in fusion, with further studies in worms, flies, zebrafish, and mice appearing to support this model. We present two in vivo assays and use a mutant V-ATPase subunit to establish that it is the H(+)-translocation/vacuole acidification function, rather than the physical presence of the V-ATPase, that promotes homotypic vacuole fusion in yeast. Furthermore, we show that acidification of the yeast vacuole in the absence of the V-ATPase rescues vacuole-fusion defects. Our results clarify the in vivo requirements of acidification for membrane fusion.

  15. Engineering hybrid exosomes by membrane fusion with liposomes

    PubMed Central

    Sato, Yuko T.; Umezaki, Kaori; Sawada, Shinichi; Mukai, Sada-atsu; Sasaki, Yoshihiro; Harada, Naozumi; Shiku, Hiroshi; Akiyoshi, Kazunari

    2016-01-01

    Exosomes are a valuable biomaterial for the development of novel nanocarriers as functionally advanced drug delivery systems. To control and modify the performance of exosomal nanocarriers, we developed hybrid exosomes by fusing their membranes with liposomes using the freeze–thaw method. Exosomes embedded with a specific membrane protein isolated from genetically modified cells were fused with various liposomes, confirming that membrane engineering methods can be combined with genetic modification techniques. Cellular uptake studies performed using the hybrid exosomes revealed that the interactions between the developed exosomes and cells could be modified by changing the lipid composition or the properties of the exogenous lipids. These results suggest that the membrane-engineering approach reported here offers a new strategy for developing rationally designed exosomes as hybrid nanocarriers for use in advanced drug delivery systems. PMID:26911358

  16. The distribution and apoptotic function of outer membrane proteins depend on mitochondrial fusion

    PubMed Central

    Weaver, David; Eisner, Verónica; Liu, Xingguo; Várnai, Péter; Hunyady, László; Gross, Atan; Hajnóczky, György

    2014-01-01

    Summary Cells deficient in mitochondrial fusion have been shown to have defects linked to the exchange of innermembrane and matrix components. Because outer-mitochondrial membrane (OMM) constituents insert directly from the cytoplasm, a role for fusion in their inter-mitochondrial transfer was unanticipated. Here we show that fibroblasts lacking the GTPases responsible for OMM fusion, Mitofusins1/2 (MFN1/2), display more heterogeneous distribution of OMM proteins. Proteins with different modes of OMM association display varying degrees of heterogeneity in Mfn1/2−/− cells and different kinetics of transfer during fusion in fusion-competent cells. Pro-apoptotic Bak exhibits marked heterogeneity, which is normalized upon expression of MFN2. Bak is critical for Bid-induced OMM permeabilization and cytochrome c release and Mfn1/2−/− cells show dysregulation of Bid-dependent apoptotic signaling. Bid sensitivity of Bak-deficient mitochondria is regained upon fusion with Bak-containing mitochondria. Thus, OMM protein distribution depends on mitochondrial fusion and is a locus of apoptotic dysfunction in conditions of fusion deficiency. PMID:24813948

  17. Lipid intermediates in membrane fusion: formation, structure, and decay of hemifusion diaphragm.

    PubMed Central

    Kozlovsky, Yonathan; Chernomordik, Leonid V; Kozlov, Michael M

    2002-01-01

    Lipid bilayer fusion is thought to involve formation of a local hemifusion connection, referred to as a fusion stalk. The subsequent fusion stages leading to the opening of a fusion pore remain unknown. The earliest fusion pore could represent a bilayer connection between the membranes and could be formed directly from the stalk. Alternatively, fusion pore can form in a single bilayer, referred to as hemifusion diaphragm (HD), generated by stalk expansion. To analyze the plausibility of stalk expansion, we studied the pathway of hemifusion theoretically, using a recently developed elastic model. We show that the stalk has a tendency to expand into an HD for lipids with sufficiently negative spontaneous splay, (~)J(s)< 0. For different experimentally relevant membrane configurations we find two characteristic values of the spontaneous splay. (~)J*(s) and (~)J**(s), determining HD dimension. The HD is predicted to have a finite equilibrium radius provided that the spontaneous splay is in the range (~)J**(s)< (~)J(s)<(~)J*(s), and to expand infinitely for (~)J(s)<(~)J**(s). In the case of common lipids, which do not fuse spontaneously, an HD forms only under action of an external force pulling the diaphragm rim apart. We calculate the dependence of the HD radius on this force. To address the mechanism of fusion pore formation, we analyze the distribution of the lateral tension emerging in the HD due to the establishment of lateral equilibrium between the deformed and relaxed portions of lipid monolayers. We show that this tension concentrates along the HD rim and reaches high values sufficient to rupture the bilayer and form the fusion pore. Our analysis supports the hypothesis that transition from a hemifusion to a fusion pore involves radial expansion of the stalk. PMID:12414697

  18. Separation of the osmotically driven fusion event from vesicle-planar membrane attachment in a model system for exocytosis

    PubMed Central

    1984-01-01

    We demonstrate that there are two experimentally distinguishable steps in the fusion of phospholipid vesicles with planar bilayer membranes. In the first step, the vesicles form a stable, tightly bound pre-fusion state with the planar membrane; divalent cations (Ca++) are required for the formation of this state if the vesicular and/or planar membrane contain negatively charged lipids. In the second step, the actual fusion of vesicular and planar membranes occurs. The driving force for this step is the osmotic swelling of vesicles attached (in the pre- fusion state) to the planar membrane. We suggest that osmotic swelling of vesicles may also be crucial for biological fusion and exocytosis. PMID:6699082

  19. High-yield membrane protein expression from E. coli using an engineered outer membrane protein F fusion.

    PubMed

    Su, Pin-Chuan; Si, William; Baker, Deidre L; Berger, Bryan W

    2013-04-01

    Obtaining high yields of membrane proteins necessary to perform detailed structural study is difficult due to poor solubility and variability in yields from heterologous expression systems. To address this issue, an Escherichia coli-based membrane protein overexpression system utilizing an engineered bacterial outer membrane protein F (pOmpF) fusion has been developed. Full-length human receptor activity-modifying protein 1 (RAMP1) was expressed using pOmpF, solubilized in FC15 and purified to homogeneity. Using circular dichroism and fluorescence spectroscopy, purified full-length RAMP1 is composed of approximately 90% α-helix, and retains its solubility and structure in FC15 over a wide range of temperatures (20-60°C). Thus, our approach provides a useful, complementary approach to achieve high-yield, full-length membrane protein overexpression for biophysical studies.

  20. Reliable titration of filamentous bacteriophages independent of pIII fusion moiety and genome size by using trypsin to restore wild-type pIII phenotype.

    PubMed

    Løset, Geir Åge; Kristinsson, Solveig G; Sandlie, Inger

    2008-04-01

    Phage display holds a key position in the use of combinatorial library approaches for the purpose of protein engineering and discovery. However, modifying the pIII protein of the phage can severely and negatively influence the infectiousness of the phage particle. This concern is particularly relevant when large pIII fusions in combination with multivalent display systems are in use. We here describe the use of trypsin to restore wild-type pIII phenotype as a small modification to the standard titration protocol. The results show that the trypsin treatment has a very large but heterogeneous effect on the phage infection efficiency, depending on the pIII fusion domain and the valence of display.

  1. Identification of a plastid protein involved in vesicle fusion and/or membrane protein translocation.

    PubMed Central

    Hugueney, P; Bouvier, F; Badillo, A; d'Harlingue, A; Kuntz, M; Camara, B

    1995-01-01

    Structural evidence has accumulated suggesting that fusion and/or translocation factors are involved in plastid membrane biogenesis. To test this hypothesis, we have developed an in vitro system in which the extent of fusion and/or translocation is monitored by the conversion of the xanthophyll epoxide (antheraxanthin) into the red ketocarotenoid (capsanthin). Only chromoplast membrane vesicles from red pepper fruits (Capsicum annuum) contain the required enzyme. Vesicles prepared from the mutant yellow cultivar are devoid of this enzyme and accumulate antheraxanthin. The fusion and/or translocation activity is characterized by complementation due to the synthesis of capsanthin and the parallel decrease of antheraxanthin when the two types of vesicles are incubated together in the presence of plastid stroma. We show that the extent of conversion is dependent upon an ATP-requiring protein that is sensitive to N-ethylmaleimide. Further purification and immunological analysis have revealed that the active factor, designated plastid fusion and/or translocation factor (Pftf), resides in a protein of 72 kDa. cDNA cloning revealed that mature Pftf has significant homology to yeast and animal (NSF) or bacterial (Ftsh) proteins involved in vesicle fusion or membrane protein translocation. Images Fig. 1 Fig. 3 Fig. 4 PMID:7777561

  2. Effect of cholesterol on the interaction of the HIV GP41 fusion peptide with model membranes. Importance of the membrane dipole potential.

    PubMed

    Buzón, Víctor; Cladera, Josep

    2006-12-26

    Fusion of viral and cell membranes is a key event in the process by which the human immunodeficiency virus (HIV) enters the target cell. Membrane fusion is facilitated by the interaction of the viral gp41 fusion peptide with the cell membrane. Using synthetic peptides and model membrane systems, it has been established that the sequence of events implies the binding of the peptide to the membrane, followed by a conformational change (transformation of unordered and helical structures into beta-aggregates) which precedes lipid mixing. It is known that this process can be influenced by the membrane lipid composition. In the present work we have undertaken a systematic study in order to determine the influence of cholesterol (abundant in the viral membrane) in the sequence of events leading to lipid mixing. Besides its effect on membrane fluidity, cholesterol can affect a less known physical parameter, the membrane dipole potential. Using the dipole potential fluorescent sensor di-8-ANEPPS together with other biophysical techniques, we show that cholesterol increases the affinity of the fusion peptide for the model membranes, and although it lowers the extent of lipid mixing, it increases the mixing rate. The influence of cholesterol on the peptide affinity and the lipid mixing rate are shown to be mainly due to its influence of the membrane dipole potential, whereas the lipid mixing extent and peptide conformational changes seem to be more dependent on other membrane parameters such as membrane fluidity and hydration.

  3. Structure of a flavivirus envelope glycoprotein in its low-pH-induced membrane fusion conformation

    PubMed Central

    Bressanelli, Stéphane; Stiasny, Karin; Allison, Steven L; Stura, Enrico A; Duquerroy, Stéphane; Lescar, Julien; Heinz, Franz X; Rey, Félix A

    2004-01-01

    Enveloped viruses enter cells via a membrane fusion reaction driven by conformational changes of specific viral envelope proteins. We report here the structure of the ectodomain of the tick-borne encephalitis virus envelope glycoprotein, E, a prototypical class II fusion protein, in its trimeric low-pH-induced conformation. We show that, in the conformational transition, the three domains of the neutral-pH form are maintained but their relative orientation is altered. Similar to the postfusion class I proteins, the subunits rearrange such that the fusion peptide loops cluster at one end of an elongated molecule and the C-terminal segments, connecting to the viral transmembrane region, run along the sides of the trimer pointing toward the fusion peptide loops. Comparison with the low-pH-induced form of the alphavirus class II fusion protein reveals striking differences at the end of the molecule bearing the fusion peptides, suggesting an important conformational effect of the missing membrane connecting segment. PMID:14963486

  4. ER network formation and membrane fusion by atlastin1/SPG3A disease variants

    PubMed Central

    Ulengin, Idil; Park, John J.; Lee, Tina H.

    2015-01-01

    At least 38 distinct missense mutations in the neuronal atlastin1/SPG3A GTPase are implicated in an autosomal dominant form of hereditary spastic paraplegia (HSP), a motor-neurological disorder manifested by lower limb weakness and spasticity and length-dependent axonopathy of corticospinal motor neurons. Because the atlastin GTPase is sufficient to catalyze membrane fusion and required to form the ER network, at least in nonneuronal cells, it is logically assumed that defects in ER membrane morphogenesis due to impaired fusion activity are the primary drivers of SPG3A-associated HSP. Here we analyzed a subset of established atlastin1/SPG3A disease variants using cell-based assays for atlastin-mediated ER network formation and biochemical assays for atlastin-catalyzed GTP hydrolysis, dimer formation, and membrane fusion. As anticipated, some variants exhibited clear deficits. Surprisingly however, at least two disease variants, one of which represents that most frequently identified in SPG3A HSP patients, displayed wild-type levels of activity in all assays. The same variants were also capable of co-redistributing ER-localized REEP1, a recently identified function of atlastins that requires its catalytic activity. Taken together, these findings indicate that a deficit in the membrane fusion activity of atlastin1 may be a key contributor, but is not required, for HSP causation. PMID:25761634

  5. Hemifusion and fusion of giant vesicles induced by reduction of inter-membrane distance

    NASA Astrophysics Data System (ADS)

    Heuvingh, J.; Pincet, F.; Cribier, S.

    2004-07-01

    Proteins involved in membrane fusion, such as SNARE or influenza virus hemagglutinin, share the common function of pulling together opposing membranes in closer contact. The reduction of inter-membrane distance can be sufficient to induce a lipid transition phase and thus fusion. We have used functionalized lipids bearing DNA bases as head groups incorporated into giant unilamellar vesicles in order to reproduce the reduction of distance between membranes and to trigger fusion in a model system. In our experiments, two vesicles were isolated and brought into adhesion by the mean of micromanipulation; their evolution was monitored by fluorescence microscopy. Actual fusion only occurred in about 5% of the experiments. In most cases, a state of “hemifusion” is observed and quantified. In this state, the outer leaflets of both vesicles' bilayers merged whereas the inner leaflets and the aqueous inner contents remained independent. The kinetics of the lipid probes redistribution is in good agreement with a diffusion model in which lipids freely diffuse at the circumference of the contact zone between the two vesicles. The minimal density of bridging structures, such as stalks, necessary to explain this redistribution kinetics can be estimated.

  6. CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization

    PubMed Central

    Jégou, Antoine; Ziyyat, Ahmed; Barraud-Lange, Virginie; Perez, Eric; Wolf, Jean Philippe; Pincet, Frédéric; Gourier, Christine

    2011-01-01

    CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm–egg fusion is a direct consequence of CD9 controlled sperm–egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs. PMID:21690351

  7. Transcytotic vesicle fusion with canalicular membranes is modulated by phospholipid species: implications for biliary lipid secretion.

    PubMed

    Hirano, N; Tazuma, S; Kajiyama, G

    1997-07-01

    Phospholipid species modulate bile metastability and the subselection of such species for biliary secretion occurs at the canalicular membrane. In this study, the role of phospholipid head groups and hydrophobic indices in transcytotic vesicle fusion with the canalicular membrane inner leaflet was investigated using rat canalicular membrane vesicles (CMV) and liposomes. The CMV were purified from Sprague-Dawley rat liver, and small unilamellar vesicles (SUV) of phosphatidylserine (PS), phosphatidylcholine (PC) and mixtures of PS/PC (1:1, 2:1 and 4:1) were labelled with 8 mol% of octadecyl rhodamine B chloride (R18). The PC species used in this study were egg yolk PC (EYPC), soybean PC (SBPC), dipalmitoyl PC (DPPC) and dilinoleoyl PC (DLPC). Fusion of SUV with CMV was initiated by the addition of a millimolar concentration of Ca2+ and the degree of fusion was estimated by the increase of R18 fluorescence. Ca(2+)-dependent fusion of SUV consisting of PS, and PS/PC (4:1) with CMV was observed (PS > PS/PC; 4:1), whereas no detectable fusion was evident between CMV and SUV of PC alone or PS/PC (1:1 or 2:1). The rank order of fusibility between CMV and SUV of PS/PC (4:1) containing various PC species was PS/DLPC > PS/SBPC > PS/EYPC > PS/DPPC. The hydrophobic index of PC as determined by high performance liquid chromatography (HPLC) was related closely to liposome fusibility (r = -0.88). These results suggest that transcytotic vesicle fusion with the canalicular membrane inner leaflet is regulated by the phospholipid hydrophobicity of the vesicles.

  8. Towards fully automated Identification of Vesicle-Membrane Fusion Events in TIRF Microscopy

    NASA Astrophysics Data System (ADS)

    Vallotton, Pascal; James, David E.; Hughes, William E.

    2007-11-01

    Total Internal Reflection Fluorescence Microscopy (TIRFM) is imposing itself as the tool of choice for studying biological activity in close proximity to the plasma membrane. For example, the exquisite selectivity of TIRFM allows monitoring the diffusion of GFP-phogrin vesicles and their recruitment to the plasma membrane in pancreatic β-cells. We present a novel computer vision system for automatically identifying the elusive fusion events of GFP-phogrin vesicles with the plasma membrane. Our method is based on robust object tracking and matched filtering. It should accelerate the quantification of TIRFM data and allow the extraction of more biological information from image data to support research in diabetes and obesity.

  9. Delivery of membrane proteins into small and giant unilamellar vesicles by charge-mediated fusion.

    PubMed

    Biner, Olivier; Schick, Thomas; Müller, Yannic; von Ballmoos, Christoph

    2016-07-01

    One of the current challenges in synthetic biology is the production of stable membrane mimetic systems and the insertion of components in these systems. Here, we employ fusion of oppositely charged liposomes to deliver separately reconstituted membrane proteins into a common lipid bilayer. After a systematic evaluation of different lipid compositions by lipid mixing and size distribution analysis, suitable conditions were further investigated for proteoliposome fusion. With this technique, we functionally coreconstituted bo3 oxidase and ATP synthase from Escherichia coli into unilamellar liposomes ranging from 100 nm to 50 μm in size. The presented method is a simple and versatile tool for oriented membrane protein reconstitution to produce biomimetic systems with increased complexity. PMID:27264202

  10. Coarse-grained molecular dynamics study of membrane fusion: Curvature effects on free energy barriers along the stalk mechanism.

    PubMed

    Kawamoto, Shuhei; Klein, Michael L; Shinoda, Wataru

    2015-12-28

    The effects of membrane curvature on the free energy barrier for membrane fusion have been investigated using coarse-grained molecular dynamics (CG-MD) simulations, assuming that fusion takes place through a stalk intermediate. Free energy barriers were estimated for stalk formation as well as for fusion pore formation using the guiding potential method. Specifically, the three different geometries of two apposed membranes were considered: vesicle-vesicle, vesicle-planar, and planar-planar membranes. The free energy barriers for the resulting fusion were found to depend importantly on the fusing membrane geometries; the lowest barrier was obtained for vesicular membranes. Further, lipid sorting was observed in fusion of the mixed membranes of dimyristoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine (DOPE). Specifically, DOPE molecules were found to assemble around the stalk to support the highly negative curved membrane surface. A consistent result for lipid sorting was observed when a simple continuum model (CM) was used, where the Helfrich energy and mixing entropy of the lipids were taken into account. However, the CM predicts a much higher free energy barrier than found using CG-MD. This discrepancy originates from the conformational changes of lipids, which were not considered in the CM. The results of the CG-MD simulations reveal that a large conformational change in the lipid takes place around the stalk region, which results in a reduction of free energy barriers along the stalk mechanism of membrane fusion.

  11. Coarse-grained molecular dynamics study of membrane fusion: Curvature effects on free energy barriers along the stalk mechanism

    SciTech Connect

    Kawamoto, Shuhei; Shinoda, Wataru; Klein, Michael L.

    2015-12-28

    The effects of membrane curvature on the free energy barrier for membrane fusion have been investigated using coarse-grained molecular dynamics (CG-MD) simulations, assuming that fusion takes place through a stalk intermediate. Free energy barriers were estimated for stalk formation as well as for fusion pore formation using the guiding potential method. Specifically, the three different geometries of two apposed membranes were considered: vesicle–vesicle, vesicle–planar, and planar–planar membranes. The free energy barriers for the resulting fusion were found to depend importantly on the fusing membrane geometries; the lowest barrier was obtained for vesicular membranes. Further, lipid sorting was observed in fusion of the mixed membranes of dimyristoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine (DOPE). Specifically, DOPE molecules were found to assemble around the stalk to support the highly negative curved membrane surface. A consistent result for lipid sorting was observed when a simple continuum model (CM) was used, where the Helfrich energy and mixing entropy of the lipids were taken into account. However, the CM predicts a much higher free energy barrier than found using CG-MD. This discrepancy originates from the conformational changes of lipids, which were not considered in the CM. The results of the CG-MD simulations reveal that a large conformational change in the lipid takes place around the stalk region, which results in a reduction of free energy barriers along the stalk mechanism of membrane fusion.

  12. Membrane skeleton restraint of surface shape change during fusion of erythrocyte membranes: evidence from use of osmotic and dielectrophoretic microforces as probes.

    PubMed Central

    Sowers, A E

    1995-01-01

    The role of the spectrin-based membrane skeleton in cell fusion was studied by following the condition-dependent diameter versus time expansion signature of the fusion zone in electrofused pairs of erythrocyte ghost membranes. Previous work showed that the presence of the dielectrophoresis-inducing alternating electric field, which is used to bring membranes into contact through pearl chain formation, had a detectable promoting effect on fusion zone expansion. Two new dielectrophoresis protocols were used in the present work to utilize this externally generated and controllable microforce field to probe the forces intrinsic to the system that drives the expansion of the fusion zone. First, fusion zones expanded to a greater diameter in a strong AC field compared to a weak AC field, and they expanded to a greater diameter if erythrocyte ghosts received a prior heat treatment (42 degrees C, 20 min). Furthermore, flat diaphragm fusion zones broke down into open lumen fusion zones sooner (i.e., had shorter lifetimes) when they were expanding more quickly. Second, changing the AC field strength at specific times during the fusion zone expansion led to an immediate visco-elastic response. However, shifting the AC field strength to zero after 5 s of fusion zone expansion resulted in a subsequent decrease in the average fusion zone diameter. This suggests not only that the spectrin-based membrane skeleton actually tends to prevent the rounding up process but that it may be capable of generating an antirounding force, which has broad implications for the role of the membrane skeleton in cell fusion. These results are consistent with the hypothesis that flat diaphragm fusion zones induced in heat-treated membranes were very easily stretched and that membrane-based forces that control or drive the expansion process must originate from membrane area that is outside rather than inside the fusion zone. Lastly, when an outward-directed osmotic pressure-based microforce was

  13. Structural basis for activation, assembly and membrane binding of ESCRT-III Snf7 filaments

    PubMed Central

    Tang, Shaogeng; Henne, W Mike; Borbat, Peter P; Buchkovich, Nicholas J; Freed, Jack H; Mao, Yuxin; Fromme, J Christopher; Emr, Scott D

    2015-01-01

    The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that catalyze multiple topologically similar membrane-remodeling processes. Although ESCRT-III subunits polymerize into spirals, how individual ESCRT-III subunits are activated and assembled together into a membrane-deforming filament remains unknown. Here, we determine X-ray crystal structures of the most abundant ESCRT-III subunit Snf7 in its active conformation. Using pulsed dipolar electron spin resonance spectroscopy (PDS), we show that Snf7 activation requires a prominent conformational rearrangement to expose protein-membrane and protein-protein interfaces. This promotes the assembly of Snf7 arrays with ~30 Å periodicity into a membrane-sculpting filament. Using a combination of biochemical and genetic approaches, both in vitro and in vivo, we demonstrate that mutations on these protein interfaces halt Snf7 assembly and block ESCRT function. The architecture of the activated and membrane-bound Snf7 polymer provides crucial insights into the spatially unique ESCRT-III-mediated membrane remodeling. DOI: http://dx.doi.org/10.7554/eLife.12548.001 PMID:26670543

  14. Crystal Structure of Dengue Virus Type 1 Envelope Protein in the Postfusion Conformation and Its Implications for Membrane Fusion

    SciTech Connect

    Nayak, Vinod; Dessau, Moshe; Kucera, Kaury; Anthony, Karen; Ledizet, Michel; Modis, Yorgo

    2009-07-31

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the 'pH sensor' that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

  15. Energetics of intermediates in membrane fusion: comparison of stalk and inverted micellar intermediate mechanisms.

    PubMed Central

    Siegel, D P

    1993-01-01

    To understand the mechanism of membrane fusion, we have to infer the sequence of structural transformations that occurs during the process. Here, it is shown how one can estimate the lipid composition-dependent free energies of intermediate structures of different geometries. One can then infer which fusion mechanism is the best explanation of observed behavior in different systems by selecting the mechanism that requires the least energy. The treatment involves no adjustable parameters. It includes contributions to the intermediate energy resulting from the presence of hydrophobic interstices within structures formed between apposed bilayers. Results of these calculations show that a modified form of the stalk mechanism proposed by others is a likely fusion mechanism in a wide range of lipid compositions, but a mechanism based on inverted micellar intermediates (IMIs) is not. This should be true even in the vicinity of the lamellar/inverted hexagonal phase transition, where IMI formation would be most facile. Another prediction of the calculations is that traces of apolar lipids (e.g., long-chain alkanes) in membranes should have a substantial influence on fusion rates in general. The same theoretical methods can be used to generate and refine mechanisms for protein-mediated fusion. PMID:8298039

  16. Membrane damage by cytolysin A-III: effects of monovalent and divalent cations

    SciTech Connect

    Liu, J.; Blumenthal, K.M.

    1987-05-01

    The effects of monovalent and divalent cations on the hemolytic activity of Cerebratulus lacteus toxin A-III were studied. In PBS buffer, A-III activity is strongly inhibited by increasing the osmotic pressure with sodium chloride but not with sucrose. Different salts, whether permeant or impermeant show qualitatively similar inhibitory effects. In low ionic strength isotonic buffer the hemolytic activity of A-III is remarkably increased, the HC50 being shifted from 2 ..mu..g/ml to 20-30 ng/ml in Hepes-sucrose. This corresponds to a 50-100 fold increase in activity. The divalent cations Zn/sup 2 +/ and Ca/sup 2 +/ also inhibit A-III activity, 0.3 mM Zn/sup 2 +/ totally abolishing A-III dependent hemolysis of human erythrocytes. Analogous studies on marker release from liposomes suggested that the effect of Zn/sup 2 +/ is due to its interaction with phospholipids. Inhibition of A-III activity by both mono and divalent cations is compatible with the importance of membrane surface potential in cytolysis. Screening of the surface potential by cations decreases membrane damage by A-III. Their data suggest a direct effect of cations on membrane phospholipid and are, to their knowledge, the first to show that cytolysin activity can be enhanced by decreasing ionic strength, in contrast to data obtained previously with other cytolysins.

  17. Autographa californica Multiple Nucleopolyhedrovirus GP64 Protein: Roles of Histidine Residues in Triggering Membrane Fusion and Fusion Pore Expansion▿†

    PubMed Central

    Li, Zhaofei; Blissard, Gary W.

    2011-01-01

    The Autographa californica multiple nucleopolyhedrovirus (AcMNPV) GP64 protein mediates membrane fusion during entry. Fusion results from a low-pH-triggered conformational change in GP64 and subsequent interactions with the membrane bilayers. The low-pH sensor and trigger of the conformational change are not known, but histidine residues are implicated because the pKa of histidine is near the threshold for triggering fusion by GP64. We used alanine substitutions to examine the roles of all individual and selected clusters of GP64 histidine residues in triggering and mediating fusion by GP64. Three histidine residues (H152, H155, and H156), located in fusion loop 2, were identified as important for membrane fusion. These three histidine residues were important for efficient pore expansion but were not required for the pH-triggered conformational change. In contrast, a cluster of three histidine residues (H245, H304, and H430) located near the base of the central coiled coil was identified as a putative sensor for low pH. Three alanine substitutions in cluster H245/H304/H430 resulted in dramatically reduced membrane fusion and the apparent loss of the prefusion conformation at neutral pH. Thus, the H245/H304/H430 cluster of histidines may function or participate as a pH sensor by stabilizing the prefusion structure of GP64. PMID:21937651

  18. Regulation of membrane fusion and secretory events in the sea urchin embryo

    SciTech Connect

    Roe, J.L.

    1990-01-01

    Membrane fusion and secretory events play a key role in fertilization and early development in the sea urchin embryo. To investigate the mechanism of membrane fusion, the effect of inhibitors of metalloendoprotease activity was studied on two model systems of cell fusion; fertilization and spiculogenesis by primary mesenchyme cells in the embryo. Both the zinc chelator, 1,10-phenanthroline, and peptide metalloprotease substrates were found to inhibit both fertilization and gamete fusion, while peptides that are not substrates of metalloproteases did not affect either process. Primary mesenchyme cells form the larval skeleton in the embryo by deposition of mineral and an organic matrix into a syncytial cavity formed by fusion of filopodia of these cells. Metalloprotease inhibitors were found to inhibit spiculogenesis both in vivo and in cultures of isolated primary mesenchyme cells, and the activity of a metalloprotease of the appropriate specificity was found in the primary mesenchyme cells. These two studies implicate the activity of a metalloprotease in a necessary step in membrane fusion. Following fertilization, exocytosis of the cortical granules results in the formation of the fertilization envelope and the hyaline layer, that surround the developing embryo. The hatching enzyme is secreted by the blastula stage sea urchin embryo, which proteolyzes the fertilization envelope surrounding the embryo, allowing the embryo to hatch. Using an assay that measures {sup 125}I-fertilization envelope degradation, the hatching enzyme was identified as a 33 kDa metalloprotease, and was purified by ion-exchange and affinity chromatography from the hatching media of Strongylocentrotus purpuratus embryos. The hatching enzyme showed a substrate preference for only a minor subset of fertilization envelope proteins.

  19. Sphingomyelin Synthase 2, but Not Sphingomyelin Synthase 1, Is Involved in HIV-1 Envelope-mediated Membrane Fusion*

    PubMed Central

    Hayashi, Yasuhiro; Nemoto-Sasaki, Yoko; Tanikawa, Takashi; Oka, Saori; Tsuchiya, Kiyoto; Zama, Kouta; Mitsutake, Susumu; Sugiura, Takayuki; Yamashita, Atsushi

    2014-01-01

    Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin, may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In this study, we examined the roles of sphingomyelin synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express Sms1 or Sms2 in Sms-deficient cells. Fusion susceptibility was ∼5-fold higher in Sms2-expressing cells (not in Sms1-expressing cells) than in Sms-deficient cells. The enhancement of fusion susceptibility observed in Sms2-expressing cells was reversed and reduced by Sms2 knockdown. We also found that catalytically nonactive Sms2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor·co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in Sms2-expressing and catalytically nonactive Sms2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in Sms2-expressing cells than Sms-deficient cells. Taken together, our research provides insight into a novel function of SMS2 which is the regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement. PMID:25231990

  20. The tail domain of tomosyn controls membrane fusion through tomosyn displacement by VAMP2

    SciTech Connect

    Yamamoto, Yasunori; Fujikura, Kohei; Sakaue, Mio; Okimura, Kenjiro; Kobayashi, Yuta; Nakamura, Toshihiro; Sakisaka, Toshiaki

    2010-08-13

    Research highlights: {yields} The tail domain of tomosyn has no effect on the tomosyn-SNARE complex formation. {yields} The tail domain binding to the VAMP-like domain allows VAMP2 to displace tomosyn. {yields} Tomosyn displacement by VAMP2 leads to SNARE complex formation. {yields} The SNARE complex formation drives membrane fusion. -- Abstract: Neurotransmitter release is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn sequesters target SNAREs (t-SNAREs) through its C-terminal VAMP-like domain (VLD). Cumulative biochemical results suggest that the tomosyn-SNARE complex is so tight that VAMP2 cannot displace tomosyn. Based on these results, the tomosyn-SNARE complex has been believed to be a dead-end complex to inhibit neurotransmitter release. On the other hand, some studies using siRNA depletion of tomosyn suggest that tomosyn positively regulates exocytosis. Therefore, it is still controversial whether tomosyn is a simple inhibitor for neurotransmitter release. We recently reported that the inhibitory activity of tomosyn is regulated by the tail domain binding to the VLD. In this study, we employed the liposome fusion assay in order to further understand modes of action of tomosyn in detail. The tail domain unexpectedly had no effect on binding of the VLD to t-SNARE-bearing liposomes. Nonetheless, the tail domain decreased the inhibitory activity of the VLD on the SNARE complex-mediated liposome fusion. These results indicate that the tail domain controls membrane fusion through tomosyn displacement by VAMP2. Deletion of the tail domain-binding region in the VLD retained the binding to t-SNAREs and promoted the liposome fusion. Together, we propose here a novel mechanism of tomosyn that controls synaptic vesicle fusion positively by serving as a placeholder for VAMP2.

  1. Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

    PubMed

    Yamada, Asako; Mitsueda, Asako; Hasan, Mahadi; Ueda, Miho; Hama, Susumu; Warashina, Shota; Nakamura, Takashi; Harashima, Hideyoshi; Kogure, Kentaro

    2016-03-01

    Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion. PMID:26667208

  2. Tri-membrane nanoparticles produced by combining liposome fusion and a novel patchwork of bicelles to overcome endosomal and nuclear membrane barriers to cargo delivery.

    PubMed

    Yamada, Asako; Mitsueda, Asako; Hasan, Mahadi; Ueda, Miho; Hama, Susumu; Warashina, Shota; Nakamura, Takashi; Harashima, Hideyoshi; Kogure, Kentaro

    2016-03-01

    Membrane fusion is a rational strategy for crossing intracellular membranes that present barriers to liposomal nanocarrier-mediated delivery of plasmid DNA into the nucleus of non-dividing cells, such as dendritic cells. Based on this strategy, we previously developed nanocarriers consisting of a nucleic acid core particle coated with four lipid membranes [Akita, et al., Biomaterials, 2009, 30, 2940-2949]. However, including the endosomal membrane and two nuclear membranes, cells possess three intracellular membranous barriers. Thus, after entering the nucleus, nanoparticles coated with four membranes would still have one lipid membrane remaining, and could impede cargo delivery. Until now, coating a core particle with an odd number of lipid membranes was challenging. To produce nanocarriers with an odd number of lipid membranes, we developed a novel coating method involving lipid nano-discs, also known as bicelles, as a material for packaging DNA in a carrier with an odd number of lipid membranes. In this procedure, bicelles fuse to form an outer coating that resembles a patchwork quilt, which allows the preparation of nanoparticles coated with only three lipid membranes. Moreover, the transfection activity of dendritic cells with these three-membrane nanoparticles was higher than that for nanoparticles coated with four lipid membranes. In summary, we developed novel nanoparticles coated with an odd number of lipid membranes using the novel "patchwork-packaging method" to deliver plasmid DNA into the nucleus via membrane fusion.

  3. Membrane-on-a-chip: microstructured silicon/silicon-dioxide chips for high-throughput screening of membrane transport and viral membrane fusion.

    PubMed

    Kusters, Ilja; van Oijen, Antoine M; Driessen, Arnold J M

    2014-04-22

    Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO2 chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers.

  4. Sequential conformational changes in the morbillivirus attachment protein initiate the membrane fusion process.

    PubMed

    Ader-Ebert, Nadine; Khosravi, Mojtaba; Herren, Michael; Avila, Mislay; Alves, Lisa; Bringolf, Fanny; Örvell, Claes; Langedijk, Johannes P; Zurbriggen, Andreas; Plemper, Richard K; Plattet, Philippe

    2015-05-01

    Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 "spacer" section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced "head-stalk" rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal "spacer" domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This "head-to-spacer" interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk's bioactivity that may prematurely activate F. Receptor-contact disrupts the "head

  5. Sequential Conformational Changes in the Morbillivirus Attachment Protein Initiate the Membrane Fusion Process

    PubMed Central

    Ader-Ebert, Nadine; Khosravi, Mojtaba; Herren, Michael; Avila, Mislay; Alves, Lisa; Bringolf, Fanny; Örvell, Claes; Langedijk, Johannes P.; Zurbriggen, Andreas; Plemper, Richard K.; Plattet, Philippe

    2015-01-01

    Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 “spacer” section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced “head-stalk” rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal “spacer” domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This “head-to-spacer” interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk’s bioactivity that may prematurely activate F. Receptor-contact disrupts the

  6. Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from the phagosomal membrane.

    PubMed

    Gallois, A; Klein, J R; Allen, L A; Jones, B D; Nauseef, W M

    2001-05-01

    Salmonella typhimurium requires a type III secretion system encoded by pathogenicity island (SPI)-2 to survive and proliferate within macrophages. This survival implies that S. typhimurium avoids or withstands bactericidal events targeted to the microbe-containing vacuole, which include intraphagosomal production of reactive oxygen species (ROS), phagosomal acidification, and delivery of hydrolytic enzymes to the phagosome via fusion with lysosomes. Recent evidence suggests that S. typhimurium alters ROS production by murine macrophages in an SPI-2-dependent manner. To gain insights into the mechanism by which S. typhimurium inhibits intraphagosomal ROS production, we analyzed the subcellular distribution of NADPH oxidase components during infection of human monocyte-derived macrophages by wild-type (WT) or several SPI-2 mutant strains of S. typhimurium. We found that the membrane component of the NADPH oxidase, flavocytochrome b(558), was actively excluded or rapidly removed from the phagosomal membrane of WT-infected monocyte-derived macrophages, thereby preventing assembly of the NADPH oxidase complex and intraphagosomal production of superoxide anion. In contrast, the NADPH oxidase assembled on and generated ROS in phagosomes containing SPI-2 mutant S. typhimurium. Subversion of NADPH oxidase assembly by S. typhimurium was accompanied by increased bacterial replication relative to that of SPI-2 mutant strains, suggesting that the ability of WT S. typhimurium to prevent NADPH oxidase assembly at the phagosomal membrane represents an important virulence factor influencing its intracellular survival.

  7. Membrane Topology of the NixA Nickel Transporter of Helicobacter pylori: Two Nickel Transport-Specific Motifs within Transmembrane Helices II and III

    PubMed Central

    Fulkerson, John F.; Mobley, Harry L. T.

    2000-01-01

    NixA, the high-affinity cytoplasmic membrane nickel transport protein of Helicobacter pylori, imports Ni2+ into the cell for insertion into the active site of the urease metalloenzyme, which is required for gastric colonization. NixA fractionates with the cytoplasmic membrane, and protein cross-linking studies suggest that NixA functions as a monomer. A preliminary topological model of NixA with seven transmembrane domains was previously proposed based on hydropathy, charge dispersion, and homology to other transporters. To test the proposed topology of NixA and relate critical residues to specific structural elements, a series of 21 NixA-LacZ and 21 NixA-PhoA fusions were created along the entire length of the protein. Expression of reporter fusions was confirmed by Western blotting with β-galactosidase- and alkaline phosphatase-specific antisera. The activities of reporter fusions near to and upstream of the predicted translational initiation demonstrated the presence of an additional amino-terminal transmembrane domain including a membrane localization signal. Activities of fusions immediately adjacent to motifs which have been shown to be requisite for Ni2+ transport localized these motifs entirely within transmembrane domains II and III. Fusion activities localized six additional Asp and Glu residues which reduced Ni2+ transport by >90% when mutated within or immediately adjacent to transmembrane domains II, V, VI, and VII. All fusions strongly support a model of NixA in which the amino and carboxy termini are located in the cytoplasm and the protein possesses eight transmembrane domains. PMID:10692379

  8. Role of a Transbilayer pH Gradient in the Membrane Fusion Activity of the Influenza Virus Hemagglutinin: Use of the R18 Assay to Monitor Membrane Merging.

    PubMed

    Ramalho-Santos, João; Pedroso De Lima, Maria C.

    1999-03-16

    It had been suggested that influenza virus-mediated membrane fusion might be dependent on a pH gradient across a target membrane. We have designed experiments in which this issue could be addressed. Two populations of liposomes were prepared, both simulating the plasma membrane of target cells, but with the pH of the internal aqueous medium buffered either at pH 7.4 (physiological cytosol pH) or at pH 5.0 (endosomal pH at which influenza virus displays maximal fusion activity). By monitoring fusion using the R18 assay, we found that the internal pH of the target liposomes did not influence membrane merging as mediated by the influenza virus hemagglutinin, thus demonstrating that a transmembrane pH gradient is not required in this fusion process.

  9. Structure and membrane remodeling activity of ESCRT-III helical polymers

    PubMed Central

    McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; Skowyra, Michael L.; Saunders, Marissa G.; Naismith, Teresa V.; Colf, Leremy A.; Afonine, Pavel; Arthur, Christopher; Sundquist, Wesley I.; Hanson, Phyllis I.; Frost, Adam

    2015-01-01

    The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 Å resolution cryo-EM reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, CHMP1B and IST1. The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecture, and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively-curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature. PMID:26634441

  10. Relaxation of Loaded ESCRT-III Spiral Springs Drives Membrane Deformation

    PubMed Central

    Chiaruttini, Nicolas; Redondo-Morata, Lorena; Colom, Adai; Humbert, Frédéric; Lenz, Martin; Scheuring, Simon; Roux, Aurélien

    2015-01-01

    Summary ESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization generates membrane curvature remains debated. Here, we show that Snf7, the main component of ESCRT-III, polymerizes into spirals at the surface of lipid bilayers. When covering the entire membrane surface, these spirals stopped growing when densely packed: they had a polygonal shape, suggesting that lateral compression could deform them. We reasoned that Snf7 spirals could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 filaments, we showed that they were deformed while growing in a confined area. Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature. This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission. PMID:26522593

  11. ESCRT-III and Vps4: a dynamic multipurpose tool for membrane budding and scission.

    PubMed

    Alonso Y Adell, Manuel; Migliano, Simona M; Teis, David

    2016-09-01

    Complex molecular machineries bud, scission and repair cellular membranes. Components of the multi-subunit endosomal sorting complex required for transport (ESCRT) machinery are enlisted when multivesicular bodies are generated, extracellular vesicles are formed, the plasma membrane needs to be repaired, enveloped viruses bud out of host cells, defective nuclear pores have to be cleared, the nuclear envelope must be resealed after mitosis and for final midbody abscission during cytokinesis. While some ESCRT components are only required for specific processes, the assembly of ESCRT-III polymers on target membranes and the action of the AAA-ATPase Vps4 are mandatory for every process. In this review, we summarize the current knowledge of structural and functional features of ESCRT-III/Vps4 assemblies in the growing pantheon of ESCRT-dependent pathways. We describe specific recruitment processes for ESCRT-III to different membranes, which could be useful to selectively inhibit ESCRT function during specific processes, while not affecting other ESCRT-dependent processes. Finally, we speculate how ESCRT-III and Vps4 might function together and highlight how the characterization of their precise spatiotemporal organization will improve our understanding of ESCRT-mediated membrane budding and scission in vivo. PMID:26910595

  12. Structure and membrane remodeling activity of ESCRT-III helical polymers.

    PubMed

    McCullough, John; Clippinger, Amy K; Talledge, Nathaniel; Skowyra, Michael L; Saunders, Marissa G; Naismith, Teresa V; Colf, Leremy A; Afonine, Pavel; Arthur, Christopher; Sundquist, Wesley I; Hanson, Phyllis I; Frost, Adam

    2015-12-18

    The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises "open" CHMP1B subunits that interlock in an elaborate domain-swapped architecture and is encircled by an outer strand of "closed" IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. Our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.

  13. Structure and membrane remodeling activity of ESCRT-III helical polymers

    DOE PAGESBeta

    McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; Skowyra, Michael L.; Saunders, Marissa G.; Naismith, Teresa V.; Colf, Leremy A.; Afonine, Pavel; Arthur, Christopher; Sundquist, Wesley I.; et al

    2015-12-18

    The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecturemore » and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. In conclusion, our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.« less

  14. Structure and membrane remodeling activity of ESCRT-III helical polymers

    SciTech Connect

    McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; Skowyra, Michael L.; Saunders, Marissa G.; Naismith, Teresa V.; Colf, Leremy A.; Afonine, Pavel; Arthur, Christopher; Sundquist, Wesley I.; Hanson, Phyllis I.; Frost, Adam

    2015-12-18

    The endosomal sorting complexes required for transport (ESCRT) proteins mediate fundamental membrane remodeling events that require stabilizing negative membrane curvature. These include endosomal intralumenal vesicle formation, HIV budding, nuclear envelope closure, and cytokinetic abscission. ESCRT-III subunits perform key roles in these processes by changing conformation and polymerizing into membrane-remodeling filaments. Here, we report the 4 angstrom resolution cryogenic electron microscopy reconstruction of a one-start, double-stranded helical copolymer composed of two different human ESCRT-III subunits, charged multivesicular body protein 1B (CHMP1B) and increased sodium tolerance 1 (IST1). The inner strand comprises “open” CHMP1B subunits that interlock in an elaborate domain-swapped architecture and is encircled by an outer strand of “closed” IST1 subunits. Unlike other ESCRT-III proteins, CHMP1B and IST1 polymers form external coats on positively curved membranes in vitro and in vivo. In conclusion, our analysis suggests how common ESCRT-III filament architectures could stabilize different degrees and directions of membrane curvature.

  15. Cell fusion in space: plasma membrane fusion in human fibroblasts during short term microgravity

    NASA Astrophysics Data System (ADS)

    Jongkind, J. F.; Visser, P.; Verkerk, A.

    During short-term microgravity in sounding rocket experiments (6 min.) the cytoskeleton undergoes changes and therefore it is possible that cell processes which are dependent on the structure and function of the cytoskeleton are influenced. A cell fusion experiment, initiated by a short electric pulse, was chosen as a model experiment for this sounding rocket experiment. Confluent monolayers of primary human skin fibroblasts, grown on coverslips, were mounted between two electrodes (distance 0.5 cm) and fused by discharging a capacitor (68 muF 250 V; 10 msec) in a low conductive medium. During a microgravity experiment in which nearly all the requirements for an optimal result were met (only the recovery of the payload was delayed) results were found that indicated that microgravity during 6 minutes did not influence cell fusion since the percentage of fused products did not change during microgravity. Within the limits of discrimination using morphological assays microgravity has no influence on the actin/cortical cytoskeleton just after electrofusion.

  16. Membrane fusion induced by the membrane mobility agent, A2C. Differentiation between fusible and non-fusible cells. Transfer of fusibility.

    PubMed

    Tavassoli, M; Kosower, N S; Halverson, C; Aoki, M; Kosower, E M

    1980-10-01

    Red cells of different species respond differently to the treatment with the membrane mobility agent, A2C, with respect to both the A2C interaction and the subsequent cell-cell interaction. Depending on whether both, one or neither of the processes are effective, some red cells (e.g., nucleated Leghorn hen red cells, rat red cells) fuse easily, some (human red cells) show morphological changes but do not fuse, and others (nucleated Rock hen red cells) show little or no response. Mixed fusion (i.e., between fusible cells of different species) is readily obtained, indicating that no species-specific recognition sites are required for A2C-induced fusion. the potential for fusion is a transferable characteristic. In the presence of fusible cells, A2C induces both heterologous and homologous fusion of otherwise 'non-fusible' cells. Electron micrographs of fusing cells after treatment with A2C reveal 'onion-ring' structures ('whorls'), free of intramembranous protein particles but different from the smooth appearance of A(2)C particles. Whorls are considered to arise from fusion-potent membrane areas. Fusion is apparent at multiple sites along the contact line between apposed membranes. The postulated appearance of vesicle-like structures along the fusion line (Kosower, E.M., Kosower, N.S. and Wegman, P. (1977) Biochim. Biophys. Acta 471, 311-329) is confirmed by micrographs. The mechanism of this fusion process is duscussed and compared to other types of fusion process. PMID:7417438

  17. Neo1 and phosphatidylethanolamine contribute to vacuole membrane fusion in Saccharomyces cerevisiae

    PubMed Central

    Wu, Yuantai; Takar, Mehmet; Cuentas-Condori, Andrea A.; Graham, Todd R.

    2016-01-01

    ABSTRACT NEO1 is an essential gene in budding yeast and belongs to a highly conserved subfamily of P-type ATPase genes that encode phospholipid flippases. Inactivation of temperature sensitive neo1ts alleles produces pleiomorphic defects in the secretory and endocytic pathways, including fragmented vacuoles. A screen for multicopy suppressors of neo1-2ts growth defects yielded YPT7, which encodes a Rab7 homolog involved in SNARE-dependent vacuolar fusion. YPT7 suppressed the vacuole fragmentation phenotype of neo1-2, but did not suppress Golgi-associated protein trafficking defects. Neo1 localizes to Golgi and endosomal membranes and was only observed in the vacuole membrane, where Ypt7 localizes, in retromer mutants or when highly overexpressed in wild-type cells. Phosphatidylethanolamine (PE) has been implicated in Ypt7-dependent vacuolar membrane fusion in vitro and is a potential transport substrate of Neo1. Strains deficient in PE synthesis (psd1Δ psd2Δ) displayed fragmented vacuoles and the neo1-2 fragmented vacuole phenotype was also suppressed by overexpression of PSD2, encoding a phosphatidylserine decarboxylase that produces PE at endosomes. In contrast, neo1-2 was not suppressed by overexpression of VPS39, an effector of Ypt7 that forms a membrane contact site potentially involved in PE transfer between vacuoles and mitochondria. These results support the crucial role of PE in vacuole membrane fusion and implicate Neo1 in concentrating PE in the cytosolic leaflet of Golgi and endosomes, and ultimately the vacuole membrane. PMID:27738552

  18. The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III.

    PubMed

    Palusińska-Szysz, Marta; Zdybicka-Barabas, Agnieszka; Reszczyńska, Emilia; Luchowski, Rafał; Kania, Magdalena; Gisch, Nicolas; Waldow, Franziska; Mak, Paweł; Danikiewicz, Witold; Gruszecki, Wiesław I; Cytryńska, Małgorzata

    2016-07-01

    Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III. PMID:27094351

  19. The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III.

    PubMed

    Palusińska-Szysz, Marta; Zdybicka-Barabas, Agnieszka; Reszczyńska, Emilia; Luchowski, Rafał; Kania, Magdalena; Gisch, Nicolas; Waldow, Franziska; Mak, Paweł; Danikiewicz, Witold; Gruszecki, Wiesław I; Cytryńska, Małgorzata

    2016-07-01

    Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III.

  20. Fusion of Legionella pneumophila outer membrane vesicles with eukaryotic membrane systems is a mechanism to deliver pathogen factors to host cell membranes.

    PubMed

    Jäger, Jens; Keese, Susanne; Roessle, Manfred; Steinert, Michael; Schromm, Andra B

    2015-05-01

    The formation and release of outer membrane vesicles (OMVs) is a phenomenon observed in many bacteria, including Legionella pneumophila. During infection, this human pathogen primarily invades alveolar macrophages and replicates within a unique membrane-bound compartment termed Legionella-containing vacuole. In the current study, we analysed the membrane architecture of L. pneumophila OMVs by small-angle X-ray scattering and biophysically characterized OMV membranes. We investigated the interaction of L. pneumophila OMVs with model membranes by Förster resonance energy transfer and Fourier transform infrared spectroscopy. These experiments demonstrated the incorporation of OMV membrane material into liposomes composed of different eukaryotic phospholipids, revealing an endogenous property of OMVs to fuse with eukaryotic membranes. Cellular co-incubation experiments showed a dose- and time-dependent binding of fluorophore-labelled OMVs to macrophages. Trypan blue quenching experiments disclosed a rapid internalization of OMVs into macrophages at 37 and 4 °C. Purified OMVs induced tumour necrosis factor-α production in human macrophages at concentrations starting at 300 ng ml(-1). Experiments on HEK293-TLR2 and TLR4/MD-2 cell lines demonstrated a dominance of TLR2-dependent signalling pathways. In summary, we demonstrate binding, internalization and biological activity of L. pneumophila OMVs on human macrophages. Our data support OMV membrane fusion as a mechanism for the remote delivery of virulence factors to host cells.

  1. Small Mismatches in Fatty Acyl Tail Lengths Can Effect Non Steroidal Anti-Inflammatory Drug Induced Membrane Fusion.

    PubMed

    Majumdar, Anupa; Sarkar, Munna

    2016-06-01

    Biological membranes are made up of a variety of lipids with diverse physicochemical properties. The lipid composition modulates different lipidic parameters, such as hydration, dynamics, lipid packing, curvature strain, etc. Changes in these parameters affect various membrane-mediated processes, such as membrane fusion which is an integral step in many biological processes. Packing defects, which originate either from mismatch in the headgroup region or in the hydrophobic acyl tail region, play a major role in modulating membrane dynamics. In this study, we demonstrate how even a small mismatch in the fatty acyl chain length, achieved by incorporation of low concentrations (up to 30 mol %) of dipalmitoylphosphatidylcholine (DPPC) into dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), alters several lipidic parameters like packing, dynamics, and headgroup hydration. This in turn affects non steroidal anti-inflammatory drug (NSAID) induced membrane fusion. Dynamic light scattering, differential scanning calorimetry, second-derivative absorption spectrophotometry, and steady-state and time-resolved fluorescence have been used to elucidate the effect of small mismatch in the tails in DMPC/DPPC mixed vesicles and how it modulates membrane fusion induced by the oxicam NSAIDs, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx). Fusion kinetics was monitored using fluorescence based fusion assays. At low DPPC concentration of 10 mol %, additional fluidization promotes lipid mixing to some extent for Mx, but at higher mol % of DPPC, subsequent increase in rigidity of membrane interior along with increase in headgroup hydration, synergistically inhibits fusion to various extents for the three different drugs, Mx, Px, and Tx.

  2. Structure and energy of fusion stalks: the role of membrane edges.

    PubMed Central

    May, Sylvio

    2002-01-01

    Fusion of lipid bilayers proceeds via a sequence of distinct structural transformations. Its early stage involves a localized, hemifused intermediate in which the proximal but not yet the distal monolayers are connected. Whereas the so-called stalk model most successfully accounts for the properties of the hemifused intermediate, there is still uncertainty about its microscopic structure and energy. We reanalyze fusion stalks using the theory of membrane elasticity. In our calculations, a short (cylindrical micelle-like) tether connects the two proximal monolayers of the hemifused membranes. The shape of the stalk and the length of the tether are calculated such as to minimize the overall free energy and to avoid the formation of voids within the hydrocarbon core. Our free energy expression is based on three internal degrees of freedom of a perturbed lipid layer: thickness, splay, and tilt deformations. Based on exactly the same model, we compare fusion stalks with and without the ability included to form sharp edges at the interfacial region between the hydrocarbon core and the polar environment. Requiring the interface to be smooth everywhere, our detailed calculations recover previous results: the stalk energies are far too high to account for the experimental observation of fusion intermediates. However, if we allow the interface to be nonsmooth, we find a remarkable reduction of the stalk free energy down to more realistic values. The corresponding structure of a nonsmooth stalk exhibits sharp edges at the transition regions between the bilayer and tether parts. In addition to that, a corner is formed at each of the two distal monolayers. We discuss the mechanism how membrane edges reduce the energy of fusion stalks. PMID:12496070

  3. Nuclear inner membrane fusion facilitated by yeast Jem1p is required for spindle pole body fusion but not for the first mitotic nuclear division during yeast mating.

    PubMed

    Nishikawa, Shuh-ichi; Hirata, Aiko; Endo, Toshiya

    2008-11-01

    During mating of budding yeast, Saccharomyces cerevisiae, two haploid nuclei fuse to produce a diploid nucleus. The process of nuclear fusion requires two J proteins, Jem1p in the endoplasmic reticulum (ER) lumen and Sec63p, which forms a complex with Sec71p and Sec72p, in the ER membrane. Zygotes of mutants defective in the functions of Jem1p or Sec63p contain two haploid nuclei that were closely apposed but failed to fuse. Here we analyzed the ultrastructure of nuclei in jem1 Delta and sec71 Delta mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 Delta and sec71 Delta mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division.

  4. Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells

    PubMed Central

    Han, Wei-Qing; Xia, Min; Xu, Ming; Boini, Krishna M.; Ritter, Joseph K.; Li, Ning-Jun; Li, Pin-Lan

    2012-01-01

    Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered in membrane raft macrodomains after Fas Ligand (FasL) stimulation as detected by confocal microscopy and membrane fraction flotation. Small-interfering RNA targeted to dysferlin prevented membrane raft clustering. Furthermore, the translocation of acid sphingomyelinase (ASMase) to membrane raft clusters, whereby local ASMase activation and ceramide production – an important step that mediates membrane raft clustering – was attenuated. Functionally, silencing of the dysferlin gene reversed FasL-induced impairment of endothelium-dependent vasodilation in isolated small coronary arteries. By monitoring fluorescence quenching or dequenching, silencing of the dysferlin gene was found to almost completely block lysosome fusion to plasma membrane upon FasL stimulation. Further studies to block C2A binding and silencing of AHNAK (a dysferlin C2A domain binding partner), showed that the dysferlin C2A domain is required for FasL-induced lysosome fusion to the cell membrane, ASMase translocation and membrane raft clustering. We conclude that dysferlin determines lysosome fusion to the plasma membrane through its C2A domain and it is therefore implicated in membrane-raft-mediated signaling and regulation of endothelial function in coronary circulation. PMID:22349696

  5. Membrane fusion promoters and inhibitors have contrasting effects on lipid bilayer structure and undulations.

    PubMed

    McIntosh, T J; Kulkarni, K G; Simon, S A

    1999-04-01

    It has been established that the fusion of both biological membranes and phospholipid bilayers can be modulated by altering their lipid composition (Chernomordik et al., 1995 .J. Membr. Biol. 146:3). In particular, when added exogenously between apposing membranes, monomyristoylphosphatidylcholine (MMPC) inhibits membrane fusion, whereas glycerol monoleate (GMO), oleic acid (OA), and arachidonic acid (AA) promote fusion. This present study uses x-ray diffraction to investigate the effects of MMPC, GMO, OA, and AA on the bending and stability of lipid bilayers when bilayers are forced together with applied osmotic pressure. The addition of 10 and 30 mol% MMPC to egg phosphatidylcholine (EPC) bilayers maintains the bilayer structure, even when the interbilayer fluid spacing is reduced to approximately 3 A, and increases the repulsive pressure between bilayers so that the fluid spacing in excess water increases by 5 and 15 A, respectively. Thus MMPC increases the undulation pressure, implying that the addition of MMPC promotes out-of-plane bending and decreases the adhesion energy between bilayers. In contrast, the addition of GMO has minor effects on the undulation pressure; 10 and 50 mol% GMO increase the fluid spacing of EPC in excess water by 0 and 2 A, respectively. However, x-ray diffraction indicates that, at small interbilayer separations, GMO, OA, or AA converts the bilayer to a structure containing hexagonally packed scattering units approximately 50 A in diameter. Thus GMO, OA, or AA destabilizes bilayer structure as apposing bilayers are brought into contact, which could contribute to their role in promoting membrane fusion.

  6. Reconstitution of membrane proteins into giant unilamellar vesicles via peptide-induced fusion.

    PubMed Central

    Kahya, N; Pécheur, E I; de Boeij, W P; Wiersma, D A; Hoekstra, D

    2001-01-01

    In this work, we present a protocol to reconstitute membrane proteins into giant unilamellar vesicles (GUV) via peptide-induced fusion. In principle, GUV provide a well-defined lipid matrix, resembling a close-to-native state for biophysical studies, including optical microspectroscopy, of transmembrane proteins at the molecular level. Furthermore, reconstitution in this manner would also eliminate potential artifacts arising from secondary interactions of proteins, when reconstituted in planar membranes supported on solid surfaces. However, assembly procedures of GUV preclude direct reconstitution. Here, for the first time, a method is described that allows the controlled incorporation of membrane proteins into GUV. We demonstrate that large unilamellar vesicles (LUV, diameter 0.1 microm), to which the small fusogenic peptide WAE has been covalently attached, readily fuse with GUV, as revealed by monitoring lipid and contents mixing by fluorescence microscopy. To monitor contents mixing, a new fluorescence-based enzymatic assay was devised. Fusion does not introduce changes in the membrane morphology, as shown by fluorescence correlation spectroscopy. Analysis of fluorescence confocal imaging intensity revealed that approximately 6 to 10 LUV fused per microm(2) of GUV surface. As a model protein, bacteriorhodopsin (BR) was reconstituted into GUV, using LUV into which BR was incorporated via detergent dialysis. BR did not affect GUV-LUV fusion and the protein was stably inserted into the GUV and functionally active. Fluorescence correlation spectroscopy experiments show that BR inserted into GUV undergoes unrestricted Brownian motion with a diffusion coefficient of 1.2 microm(2)/s. The current procedure offers new opportunities to address issues related to membrane-protein structure and dynamics in a close-to-native state. PMID:11509360

  7. Identification of viral membrane proteins required for cell fusion and viral dissemination that are modified during vaccinia virus persistence.

    PubMed

    Ortiz, M A; Paez, E

    1994-01-01

    position of the additional mutation coincided with a small deletion occurring in the HindIII F fragment. This deletion affected the 5'-end and promoter sequences of the 37 kDa envelope protein gene and produced a lack of expression of this protein, shown by others to be involved in the formation of extracellular enveloped virus in infected cells. These results shown that viruses with mutations of viral membrane proteins required for cell fusion and viral dissemination are selected during vaccinia virus persistence in cell culture.

  8. The cesium-induced delay in myoblast membrane fusion is accompanied by changes in cellular subfraction lipid composition.

    PubMed

    Santini, M T; Indovina, P L; Cantafora, A

    1991-11-18

    We have recently demonstrated that the delay in myoblast membrane fusion induced by cesium is accompanied by changes in isolated membrane lipids (Santini, M.T., Indovina, P.L., Cantafora, A. and Blotta, I. (1990) Biochim. Biophys. Acta 1023, 298-304). In the present study, we have investigated changes in the lipid profile of total cell homogenates and microsomal membrane fractions during myoblast membrane fusion as well as the effects that addition of cesium may have on these lipid variations in order to try to understand the production and translocation of lipids during this myogenic process. The data presented here indicate that the lipid composition of cell homogenates and microsomes varies in a different manner from isolated plasma membranes during myogenic fusion. In addition, cesium affects, in a different manner, the normally-occurring lipid production and distribution which takes place in each subcellular fraction.

  9. A soluble form of Epstein-Barr virus gH/gL inhibits EBV-induced membrane fusion and does not function in fusion

    SciTech Connect

    Rowe, Cynthia L.; Connolly, Sarah A.; Chen, Jia; Jardetzky, Theodore S.; Longnecker, Richard

    2013-02-05

    We investigated whether soluble EBV gH/gL (sgH/gL) functions in fusion and made a series of truncations of gH/gL domains based on the gH/gL crystal structure. We found sgH/gL failed to mediate cell-cell fusion both when co-expressed with the other entry glycoproteins and when added exogenously to fusion assays. Interestingly, sgH/gL inhibited cell-cell fusion in a dose dependent manner when co-expressed. sgH/gL from HSV was unable to inhibit EBV fusion, suggesting the inhibition was specific to EBV gH/gL. sgH/gL stably binds gp42, but not gB nor gH/gL. The domain mutants, DI/gL, DI-II/gL and DI-II-III/gL were unable to bind gp42. Instead, DI-II/gL, DI-II-III/gL and sgH/gL but not DI/gL decreased the expression of gp42, resulting in decreased overall fusion. Overall, our results suggest that domain IV may be required for proper folding and the transmembrane domain and cytoplasmic tail of EBV gH/gL are required for the most efficient fusion.

  10. The BAR domain proteins: molding membranes in fission, fusion, and phagy.

    PubMed

    Ren, Gang; Vajjhala, Parimala; Lee, Janet S; Winsor, Barbara; Munn, Alan L

    2006-03-01

    The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt alpha-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. PMID:16524918

  11. The BAR Domain Proteins: Molding Membranes in Fission, Fusion, and Phagy

    PubMed Central

    Ren, Gang; Vajjhala, Parimala; Lee, Janet S.; Winsor, Barbara; Munn, Alan L.

    2006-01-01

    The Bin1/amphiphysin/Rvs167 (BAR) domain proteins are a ubiquitous protein family. Genes encoding members of this family have not yet been found in the genomes of prokaryotes, but within eukaryotes, BAR domain proteins are found universally from unicellular eukaryotes such as yeast through to plants, insects, and vertebrates. BAR domain proteins share an N-terminal BAR domain with a high propensity to adopt α-helical structure and engage in coiled-coil interactions with other proteins. BAR domain proteins are implicated in processes as fundamental and diverse as fission of synaptic vesicles, cell polarity, endocytosis, regulation of the actin cytoskeleton, transcriptional repression, cell-cell fusion, signal transduction, apoptosis, secretory vesicle fusion, excitation-contraction coupling, learning and memory, tissue differentiation, ion flux across membranes, and tumor suppression. What has been lacking is a molecular understanding of the role of the BAR domain protein in each process. The three-dimensional structure of the BAR domain has now been determined and valuable insight has been gained in understanding the interactions of BAR domains with membranes. The cellular roles of BAR domain proteins, characterized over the past decade in cells as distinct as yeasts, neurons, and myocytes, can now be understood in terms of a fundamental molecular function of all BAR domain proteins: to sense membrane curvature, to bind GTPases, and to mold a diversity of cellular membranes. PMID:16524918

  12. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed Central

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature. PMID:27239276

  13. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication?

    PubMed

    Imhof, Simon; Fragoso, Cristina; Hemphill, Andrew; von Schubert, Conrad; Li, Dong; Legant, Wesley; Betzig, Eric; Roditi, Isabel

    2016-01-01

    Diverse structures facilitate direct exchange of proteins between cells, including plasmadesmata in plants and tunnelling nanotubes in bacteria and higher eukaryotes.  Here we describe a new mechanism of protein transfer, flagellar membrane fusion, in the unicellular parasite Trypanosoma brucei. When fluorescently tagged trypanosomes were co-cultured, a small proportion of double-positive cells were observed. The formation of double-positive cells was dependent on the presence of extracellular calcium and was enhanced by placing cells in medium supplemented with fresh bovine serum. Time-lapse microscopy revealed that double-positive cells arose by bidirectional protein exchange in the absence of nuclear transfer.  Furthermore, super-resolution microscopy showed that this process occurred in ≤1 minute, the limit of temporal resolution in these experiments. Both cytoplasmic and membrane proteins could be transferred provided they gained access to the flagellum. Intriguingly, a component of the RNAi machinery (Argonaute) was able to move between cells, raising the possibility that small interfering RNAs are transported as cargo. Transmission electron microscopy showed that shared flagella contained two axonemes and two paraflagellar rods bounded by a single membrane. In some cases flagellar fusion was partial and interactions between cells were transient. In other cases fusion occurred along the entire length of the flagellum, was stable for several hours and might be irreversible. Fusion did not appear to be deleterious for cell function: paired cells were motile and could give rise to progeny while fused. The motile flagella of unicellular organisms are related to the sensory cilia of higher eukaryotes, raising the possibility that protein transfer between cells via cilia or flagella occurs more widely in nature.

  14. The outer membrane phospholipase A is essential for membrane integrity and type III secretion in Shigella flexneri

    PubMed Central

    Wang, Xia; Jiang, Feng; Zheng, Jianhua; Chen, Lihong; Dong, Jie; Sun, Lilian; Zhu, Yafang; Liu, Bo; Yang, Jian; Yang, Guowei

    2016-01-01

    Outer membrane phospholipase A (OMPLA) is an enzyme located in the outer membrane of Gram-negative bacteria. OMPLA exhibits broad substrate specificity, and some of its substrates are located in the cellular envelope. Generally, the enzymatic activity can only be induced by perturbation of the cell envelope integrity through diverse methods. Although OMPLA has been thoroughly studied as a membrane protein in Escherichia coli and is constitutively expressed in many other bacterial pathogens, little is known regarding the functions of OMPLA during the process of bacterial infection. In this study, the proteomic and transcriptomic data indicated that OMPLA in Shigella flexneri, termed PldA, both stabilizes the bacterial membrane and is involved in bacterial infection under ordinary culture conditions. A series of physiological assays substantiated the disorganization of the bacterial outer membrane and the periplasmic space in the ΔpldA mutant strain. Furthermore, the ΔpldA mutant strain showed decreased levels of type III secretion system expression, contributing to the reduced internalization efficiency in host cells. The results of this study support that PldA, which is widespread across Gram-negative bacteria, is an important factor for the bacterial life cycle, particularly in human pathogens. PMID:27655730

  15. Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events

    PubMed Central

    Terasaki, Mark; Miyake, Katsuya; McNeil, Paul L.

    1997-01-01

    A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ∼40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (∼5–10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ∼3 mM Ca2+ (SW is ∼10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion

  16. Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells

    PubMed Central

    Hissa, Bárbara; Duarte, Jacqueline G.; Kelles, Ludmila F.; Santos, Fabio P.; del Puerto, Helen L.; Gazzinelli-Guimarães, Pedro H.; de Paula, Ana M.; Agero, Ubirajara; Mesquita, Oscar N.; Guatimosim, Cristina; Chiari, Egler; Andrade, Luciana O.

    2012-01-01

    Background Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages) and non-professional (epithelial) phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. Methodology/Principal Finding In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. Conclusion/Significance Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of lysosomes are

  17. Free Energy Landscape of Rim-Pore Expansion in Membrane Fusion

    PubMed Central

    Risselada, Herre Jelger; Smirnova, Yuliya; Grubmüller, Helmut

    2014-01-01

    The productive fusion pore in membrane fusion is generally thought to be toroidally shaped. Theoretical studies and recent experiments suggest that its formation, in some scenarios, may be preceded by an initial pore formed near the rim of the extended hemifusion diaphragm (HD), a rim-pore. This rim-pore is characterized by a nontoroidal shape that changes with size. To determine this shape as well as the free energy along the pathway of rim-pore expansion, we derived a simple analytical free energy model. We argue that dilation of HD material via expansion of a rim-pore is favored over a regular, circular pore. Further, the expanding rim-pore faces a free energy barrier that linearly increases with HD size. In contrast, the tension required to expand the rim-pore decreases with HD size. Pore flickering, followed by sudden opening, occurs when the tension in the HD competes with the line energy of the rim-pore, and the rim-pore reaches its equilibrium size before reaching the critical pore size. The experimental observation of flickering and closing fusion pores (kiss-and-run) is very well explained by the observed behavior of rim-pores. Finally, the free energy landscape of rim-pore expansion/HD dilation may very well explain why some cellular fusion reactions, in their attempt to minimize energetic costs, progress via alternative formation and dilation of microscopic hemifusion intermediates. PMID:25418297

  18. Structure and Dynamics of a Fusion Peptide Helical Hairpin on the Membrane Surface: Comparison of Molecular Simulations and NMR

    PubMed Central

    2015-01-01

    The conserved N-terminal residues of the HA2 subunit of influenza hemagglutinin (fusion peptide) are essential for membrane fusion and viral entry. Recent NMR studies showed that the 23-residue fusion peptide forms a helical hairpin that undergoes rocking motion relative to the membrane surface on a nanosecond time scale. To compare with NMR and to obtain a detailed molecular picture of the peptide–membrane interaction, we performed molecular dynamics simulations of the fusion peptide in explicit dimyristoylphosphatidylcholine and with the IMM1 implicit membrane model. To account for low and neutral pH conditions, simulations were performed with acidic groups (E11 and D19) protonated and unprotonated, respectively. The hairpin structure was stable in the simulations, with the N-terminal helix buried more deeply into the hydrophobic membrane interior than the C-terminal helix. Interactions between the tryptophans in the fusion peptide and phospholipid residues contribute to peptide orientation. Higher flexibility of the hairpin was observed in the implicit membrane simulations. Internal correlation functions of backbone N–H vectors were fit to the extended Lipari–Szabo model-free approach to obtain order parameters and correlation times. Good agreement with the NMR results was obtained for orientational fluctuations around the hairpin axis (rotation), but those around the perpendicular axis (tilting) were more limited in the simulations than inferred from the NMR experiments. PMID:24712538

  19. Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

    PubMed

    Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

    2014-11-01

    Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect.

  20. Membrane Fusion Mediated by pH-Low-Insertion-Peptide (pHLIP)

    NASA Astrophysics Data System (ADS)

    Daniels, Jennifer; Yao, Lan; Engelman, Donald; Andreev, Oleg; Reshetnyak, Yana

    2012-02-01

    Liposomes are traditionally used as drug delivery carriers. The major mechanism of liposome entry into cell is endocytotic. First, the endocytotic pathway of cellular entry is non-specific: the delivery of therapeutics occurs to cells in both diseased and healthy tissues. Second, liposomes are usually trapped in endosome/lysosome, which prevents delivery of therapeutics to cytoplasm. We proposed to use pHLIP (pH-Low-Insertion-Peptide) to promote selective delivery of the liposome content to cytoplasm of cancer cells. We showed that liposomes coated with PEG polymer and pHLIP peptide enhance membrane fusion in acidic environments. pHLIP promotes fusion between lipid bilayer of liposome and plasma membrane or membrane of endosome/lysosome, which results in intracellular delivery of payload. Liposomes composed of 5 % pHLIP and 5 % PEG were ideal for the delivery. Since cancer and other pathological states produce an acid extracellular environment, this allows the liposome to target diseased tissue while avoiding healthy tissue (with neutral pH in extracellular space). The work is supported by NIH grants CA133890 to OAA, DME, YRK.

  1. Proteasome Impairment Induces Recovery of Mitochondrial Membrane Potential and an Alternative Pathway of Mitochondrial Fusion

    PubMed Central

    Shirozu, Ryohei; Yashiroda, Hideki

    2015-01-01

    Mitochondria are vital and highly dynamic organelles that continuously fuse and divide to maintain mitochondrial quality. Mitochondrial dysfunction impairs cellular integrity and is known to be associated with various human diseases. However, the mechanism by which the quality of mitochondria is maintained remains largely unexplored. Here we show that impaired proteasome function recovers the growth of yeast cells lacking Fzo1, a pivotal protein for mitochondrial fusion. Decreased proteasome activity increased the mitochondrial oxidoreductase protein Mia40 and the ratio of the short isoform of mitochondrial intermembrane protein Mgm1 (s-Mgm1) to the long isoform (l-Mgm1). The increase in Mia40 restored mitochondrial membrane potential, while the increase in the s-Mgm1/l-Mgm1 ratio promoted mitochondrial fusion in an Fzo1-independent manner. Our findings demonstrate a new pathway for mitochondrial quality control that is induced by proteasome impairment. PMID:26552703

  2. Biochemical requirements for the targeting and fusion of ER-derived transport vesicles with purified yeast Golgi membranes

    PubMed Central

    1996-01-01

    In order for secretion to progress, ER-derived transport vesicles must target to, and fuse with the cis-Golgi compartment. These processes have been reconstituted using highly enriched membrane fractions and partially purified soluble components. The functionally active yeast Golgi membranes that have been purified are highly enriched in the cis- Golgi marker enzymes alpha 1,6 mannosyltransferase and GDPase. Fusion of transport vesicles with these membranes requires both GTP and ATP hydrolysis, and depends on cytosolic and peripheral membrane proteins. At least two protein fractions from yeast cytosol are required for the reconstitution of ER-derived vesicle fusion. Soluble fractions prepared from temperature-sensitive mutants revealed requirements for the Ypt1p, Sec19p, Sly1p, Sec7p, and Uso1 proteins. A model for the sequential involvement of these components in the targeting and fusion reaction is proposed. PMID:8636207

  3. Mutational analysis of the vesicular stomatitis virus glycoprotein G for membrane fusion domains.

    PubMed

    Li, Y; Drone, C; Sat, E; Ghosh, H P

    1993-07-01

    The spike glycoprotein G of vesicular stomatitis virus (VSV) induces membrane fusion at low pH. We used linker insertion mutagenesis to characterize the domain(s) of G glycoprotein involved in low-pH-induced membrane fusion. Two or three amino acids were inserted in frame into various positions in the extracellular domain of G, and 14 mutants were isolated. All of the mutants expressed fully glycosylated proteins in COS cells. However, only seven mutant G glycoproteins were transported to the cell surface. Two of these mutants, D1 and A6, showed wild-type fusogenic properties. The mutant A2 had a temperature-sensitive defect in the transport of the mutant G glycoprotein to the cell surface. The other four mutants, H2, H5, H10, and A4, although present in cell surface, failed to induce cell fusion when cells expressing these mutant glycoproteins were exposed to acidic pH. These four mutant G proteins could form trimers, indicating that the defect in fusion was not due to defective oligomerization. One of these mutations, H2, is within a region of conserved, uncharged amino acids that has been proposed as a possible fusogenic sequence. The mutation in H5 was about 70 amino acids downstream of the mutation in H2, while mutations in H10 and A4 were about 300 amino acids downstream of the mutation in H2. Conserved sequences were also noted in the H10 and A4 segment. The results suggest that in the case of VSV G glycoprotein, the fusogenic activity may involve several spatially separated regions in the extracellular domain of the protein.

  4. Interaction between the Hemagglutinin-Neuraminidase and Fusion Glycoproteins of Human Parainfluenza Virus Type III Regulates Viral Growth In Vivo

    PubMed Central

    Xu, Rui; Palmer, Samantha G.; Porotto, Matteo; Palermo, Laura M.; Niewiesk, Stefan; Wilson, Ian A.; Moscona, Anne

    2013-01-01

    ABSTRACT Paramyxoviruses, enveloped RNA viruses that include human parainfluenza virus type 3 (HPIV3), cause the majority of childhood viral pneumonia. HPIV3 infection starts when the viral receptor-binding protein engages sialic acid receptors in the lung and the viral envelope fuses with the target cell membrane. Fusion/entry requires interaction between two viral surface glycoproteins: tetrameric hemagglutinin-neuraminidase (HN) and fusion protein (F). In this report, we define structural correlates of the HN features that permit infection in vivo. We have shown that viruses with an HN-F that promotes growth in cultured immortalized cells are impaired in differentiated human airway epithelial cell cultures (HAE) and in vivo and evolve in HAE into viable viruses with less fusogenic HN-F. In this report, we identify specific structural features of the HN dimer interface that modulate HN-F interaction and fusion triggering and directly impact infection. Crystal structures of HN, which promotes viral growth in vivo, show a diminished interface in the HN dimer compared to the reference strain’s HN, consistent with biochemical and biological data indicating decreased dimerization and decreased interaction with F protein. The crystallographic data suggest a structural explanation for the HN’s altered ability to activate F and reveal properties that are critical for infection in vivo. IMPORTANCE Human parainfluenza viruses cause the majority of childhood cases of croup, bronchiolitis, and pneumonia worldwide. Enveloped viruses must fuse their membranes with the target cell membranes in order to initiate infection. Parainfluenza fusion proceeds via a multistep reaction orchestrated by the two glycoproteins that make up its fusion machine. In vivo, viruses adapt for survival by evolving to acquire a set of fusion machinery features that provide key clues about requirements for infection in human beings. Infection of the lung by parainfluenzavirus is determined by

  5. Movement of a fluorescent lipid label from a labeled erythrocyte membrane to an unlabeled erythrocyte membrane following electric-field-induced fusion.

    PubMed Central

    Sowers, A E

    1985-01-01

    A short burst of electric field pulses was used to induce nearly simultaneous fusion among 50% or more of a population composed of unlabeled erythrocytes and erythrocytes labeled with the fluorescent lipid analogue DiI (1,1'-dihexadecyl-3,3,3'',3'-tetra-methylindo carbocyanine perchlorate). Fusion products that ended in an hourglass shape were selected for analysis. The net movement of the label from the labeled membrane to the adjacent unlabeled membrane in each of the hourglass-shaped fusion products was recorded by micrography at various known times after the fusion took place, but before equilibrium was achieved. The lateral concentration gradients were measured by densitometry and compared with predictions based on Huang's model (Huang, H.-W., 1973, J. Theor. Biol., 40:11-17) for lateral diffusion on a spherical membrane. The average lateral diffusion coefficients, 3.8 and 8.1 X 10(-9) cm2/s in pH 7.4 isotonic phosphate buffer at 23-25 degrees C and 35-37 degrees C, respectively, compare very favorably with the results of three published photobleaching studies of the lateral diffusion of DiI in erythrocyte membranes. While the fusion approach to measuring lateral diffusion is not new, it has not enjoyed widespread use because of the uncertainty in the degree of fusion synchrony and low fusion yield. This study shows that the use of pulsed electric fields to induce synchronous fusion is a promising approach to overcome both of these drawbacks and yield results comparable to those obtainable by the photobleaching approach. Images FIGURE 2 PMID:3986281

  6. Insulin-stimulated plasma membrane fusion of Glut4 glucose transporter-containing vesicles is regulated by phospholipase D1.

    PubMed

    Huang, Ping; Altshuller, Yelena M; Hou, June Chunqiu; Pessin, Jeffrey E; Frohman, Michael A

    2005-06-01

    Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insulin signaling, and traffics with Glut4 to exocytic sites. Increasing PLD1 activity facilitates glucose uptake, whereas decreasing PLD1 activity is inhibitory. Diminished PA production does not substantially hinder trafficking of the vesicles or their docking at the plasma membrane, but it does impede fusion-mediated extracellular exposure of the transporter. The fusion block caused by RNA interference-mediated PLD1 deficiency is rescued by exogenous provision of a lipid that promotes fusion pore formation and expansion, suggesting that the step regulated by PA is late in the process of vesicle fusion. PMID:15772157

  7. SNARE Molecules in Marchantia polymorpha: Unique and Conserved Features of the Membrane Fusion Machinery.

    PubMed

    Kanazawa, Takehiko; Era, Atsuko; Minamino, Naoki; Shikano, Yu; Fujimoto, Masaru; Uemura, Tomohiro; Nishihama, Ryuichi; Yamato, Katsuyuki T; Ishizaki, Kimitsune; Nishiyama, Tomoaki; Kohchi, Takayuki; Nakano, Akihiko; Ueda, Takashi

    2016-02-01

    The membrane trafficking pathway has been diversified in a specific way for each eukaryotic lineage, probably to fulfill specific functions in the organisms. In green plants, comparative genomics has supported the possibility that terrestrialization and/or multicellularization could be associated with the elaboration and diversification of membrane trafficking pathways, which have been accomplished by an expansion of the numbers of genes required for machinery components of membrane trafficking, including soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, information regarding membrane trafficking pathways in basal land plant lineages remains limited. In the present study, we conducted extensive analyses of SNARE molecules, which mediate membrane fusion between target membranes and transport vesicles or donor organelles, in the liverwort, Marchantia polymorpha. The M. polymorpha genome contained at least 34 genes for 36 SNARE proteins, comprising fundamental sets of SNARE proteins that are shared among land plant lineages with low degrees of redundancy. We examined the subcellular distribution of a major portion of these SNARE proteins by expressing Citrine-tagged SNARE proteins in M. polymorpha, and the results showed that some of the SNARE proteins were targeted to different compartments from their orthologous products in Arabidopsis thaliana. For example, MpSYP12B was localized to the surface of the oil body, which is a unique organelle in liverworts. Furthermore, we identified three VAMP72 members with distinctive structural characteristics, whose N-terminal extensions contain consensus sequences for N-myristoylation. These results suggest that M. polymorpha has acquired unique membrane trafficking pathways associated with newly acquired machinery components during evolution. PMID:26019268

  8. Cloning of the outer membrane high-affinity Fe(III)-pyochelin receptor of Pseudomonas aeruginosa.

    PubMed Central

    Ankenbauer, R G

    1992-01-01

    Pseudomonas aeruginosa produces the phenolic siderophore pyochelin under iron-limiting conditions. In this study, an Fe(III)-pyochelin transport-negative (Fpt-) strain, IA613, was isolated and characterized. 55Fe(III)-pyochelin transport assays determined that no Fe(III)-pyochelin associated with the Fpt- IA613 cells while a significant amount associated with KCN-poisoned Fpt+ cells. A P. aeruginosa genomic library was constructed in the IncP cosmid pLAFR1. The genomic library was mobilized into IA613, and a recombinant cosmid, pCC41, which complemented the Fpt- phenotype of IA613, was isolated. pCC41 contained a 28-kb insert of P. aeruginosa DNA, and the Fpt(-)-complementing region was localized to a 3.6-kb BamHI-EcoRI fragment by deletion and subcloning of the insert. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of IA613 revealed that it lacked a 75-kDa outer membrane protein present in Fpt+ strains. IA613 strains bearing plasmid pRML303, which carries the 3.6-kb BamHI-EcoRI fragment of pCC41, expressed the 75-kDa outer membrane protein and demonstrated a 55Fe(III)-pyochelin transport phenotype identical to that of a wild-type Fpt+ strain. Minicell analysis demonstrated that the 3.6-kb BamHI-EcoRI fragment of pCC41 encoded a protein of approximately 75 kDa. The results presented here and in a previous report (D. E. Heinrichs, L. Young, and K. Poole, Infect. Immun. 59:3680-3684, 1991) lead to the conclusion that the 75-kDa outer membrane protein is the high-affinity receptor for Fe(III)-pyochelin in P. aeruginosa. Images PMID:1320609

  9. Study of Cobalt(III) Corrole as the Neutral Ionophore for Nitrite and Nitrate Detection via Polymeric Membrane Electrodes.

    PubMed

    Yang, Si; Meyerhoff, Mark E

    2013-12-01

    Cobalt(III) 5, 10, 15-tris(4-tert-butylphenyl) corrole was synthesized and incorporated into plasticized poly(vinyl chloride) membranes and studied as a neutral carrier ionophore via potentiometry. This cobalt(III) complex has binding affinity to nitrite, and the resulting membrane electrode yields reversible and Nernstian response toward nitrite. Enhanced nitrite selectivity is observed over other anions, including lipophilic anions such as thiocyanate and perchlorate when an appropriate amount of lipophilic cationic sites are added to the membrane phase. Detection limit to nitrite is ca. 5 µM. Using tributylphosphate as the plasticizer with the cobalt(III) corrole species yields electrodes with enhanced nitrate selectivity.

  10. Effects of chronic treatment with sodium tetrachloroaurate(III) in mice and membrane models.

    PubMed

    Suwalsky, Mario; Zambenedetti, Pamela; Carpené, Emilio; Ibnlkayat, Meryem; Wittkowski, Werner; Messori, Luigi; Zatta, Paolo

    2004-12-01

    Gold is a nonessential element with a variety of applications in medicine. A few gold(I) compounds are used in the clinics for treatment of rheumatoid arthritis and of discoid lupus. Some novel gold(III) compounds are under evaluation as anticancer agents. It is known that gold compounds generally produce toxic effects on the kidneys and characteristic lesions in the brain. However, information concerning the neurotoxicity of gold derivatives in humans as well as in experimental toxicology is rather scarce. For this reason we tried to shed some further light on this aspect of gold neurotoxicity by chronic treatment of mice with sodium tetrachloroaurate(III) in order to observe possible biophysical and morphological alterations that may occur in the brain. Chronic gold treatment resulted in a markedly decreased expression of metallothioneins and of glial fibrillary acidic protein in astrocytes of different brain areas. To examine its effects on cell membranes, interactions of sodium tetrachloroaurate(III) with molecular models were also evaluated. The models consisted in bilayers built-up of classes of phospholipids located in the outer and inner monolayers of biological membranes. Structural perturbation of cell membrane models was observed only at concentrations 10(5) times higher than those detected in the brains of animals after three months' treatment. These results show that toxic effects on animal brain upon treatment with sodium tetrachloroaurate develop with difficulty and may be observed only at high doses. PMID:15541497

  11. The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts).

    PubMed

    Maurice, Pascal; Waeckel, Ludovic; Pires, Viviane; Sonnet, Pascal; Lemesle, Monique; Arbeille, Brigitte; Vassy, Jany; Rochette, Jacques; Legrand, Chantal; Fauvel-Lafève, Françoise

    2006-04-01

    Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and alpha2beta1 integrin) for efficient platelet activation. PMID:16205938

  12. Physiological lipid composition is vital for homotypic ER membrane fusion mediated by the dynamin-related GTPase Sey1p

    PubMed Central

    Sugiura, Shintaro; Mima, Joji

    2016-01-01

    Homotypic fusion of the endoplasmic reticulum (ER) is required for generating and maintaining the characteristic reticular ER membrane structures. This organelle membrane fusion process depends on the ER-bound dynamin-related GTPases, such as atlastins in animals and Sey1p in yeast. Here, to investigate whether specific lipid molecules facilitate GTPase-dependent ER membrane fusion directly, we comprehensively evaluated membrane docking and lipid mixing of reconstituted proteoliposomes bearing purified Sey1p and a set of ER-mimicking lipids, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, and ergosterol. Remarkably, we revealed that each specific lipid species contributed little to membrane docking mediated by Sey1p. Nevertheless, Sey1p-dependent lipid mixing was strongly reduced by omitting three major acidic lipids from the ER-mimicking set and, moreover, was entirely abolished by omitting either phosphatidylethanolamine or ergosterol. Our reconstitution studies thus established that physiological lipid composition is vital for lipid bilayer rearrangements in GTPase-mediated homotypic ER membrane fusion. PMID:26838333

  13. Transmembrane Peptides Stabilize Inverted Cubic Phases in a Biphasic Length-Dependent Manner: Implications for Protein-Induced Membrane Fusion

    PubMed Central

    Siegel, D. P.; Cherezov, V.; Greathouse, D. V.; Koeppe, R. E.; Killian, J. Antoinette; Caffrey, M.

    2006-01-01

    WALP peptides consist of repeating alanine-leucine sequences of different lengths, flanked with tryptophan “anchors” at each end. They form membrane-spanning α-helices in lipid membranes, and mimic protein transmembrane domains. WALP peptides of increasing length, from 19 to 31 amino acids, were incorporated into N-monomethylated dioleoylphosphatidylethanolamine (DOPE-Me) at concentrations up to 0.5 mol % peptide. When pure DOPE-Me is heated slowly, the lamellar liquid crystalline (Lα) phase first forms an inverted cubic (QII) phase, and the inverted hexagonal (HII) phase at higher temperatures. Using time-resolved x-ray diffraction and slow temperature scans (1.5°C/h), WALP peptides were shown to decrease the temperatures of QII and HII phase formation (TQ and TH, respectively) as a function of peptide concentration. The shortest and longest peptides reduced TQ the most, whereas intermediate lengths had weaker effects. These findings are relevant to membrane fusion because the first step in the Lα/QII phase transition is believed to be the formation of fusion pores between pure lipid membranes. These results imply that physiologically relevant concentrations of these peptides could increase the susceptibility of biomembrane lipids to fusion through an effect on lipid phase behavior, and may explain one role of the membrane-spanning domains in the proteins that mediate membrane fusion. PMID:16214859

  14. Ferlins: regulators of vesicle fusion for auditory neurotransmission, receptor trafficking and membrane repair.

    PubMed

    Lek, Angela; Evesson, Frances J; Sutton, R Bryan; North, Kathryn N; Cooper, Sandra T

    2012-02-01

    Ferlins are a family of multiple C2 domain proteins with emerging roles in vesicle fusion and membrane trafficking. Ferlin mutations are associated with muscular dystrophy (dysferlin) and deafness (otoferlin) in humans, and infertility in Caenorhabditis elegans (Fer-1) and Drosophila (misfire), demonstrating their importance for normal cellular functioning. Ferlins show ancient origins in eukaryotic evolution and are detected in all eukaryotic kingdoms, including unicellular eukaryotes and apicomplexian protists, suggesting origins in a common ancestor predating eukaryotic evolutionary branching. The characteristic feature of the ferlin family is their multiple tandem cytosolic C2 domains (five to seven C2 domains), the most of any protein family, and an extremely rare feature amongst eukaryotic proteins. Ferlins also bear a unique nested DysF domain and small conserved 60-70 residue ferlin-specific sequences (Fer domains). Ferlins segregate into two subtypes based on the presence (type I ferlin) or absence (type II ferlin) of the DysF and FerA domains. Ferlins have diverse tissue-specific and developmental expression patterns, with ferlin animal models united by pathologies arising from defects in vesicle fusion. Consistent with their proposed role in vesicle trafficking, ferlin interaction partners include cytoskeletal motors, other vesicle-associated trafficking proteins and transmembrane receptors or channels. Herein we summarize the research history of the ferlins, an intriguing family of structurally conserved proteins with a preserved ancestral function as regulators of vesicle fusion and receptor trafficking.

  15. HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth.

    PubMed

    Blondelle, Jordan; Ohno, Yusuke; Gache, Vincent; Guyot, Stéphane; Storck, Sébastien; Blanchard-Gutton, Nicolas; Barthélémy, Inès; Walmsley, Gemma; Rahier, Anaëlle; Gadin, Stéphanie; Maurer, Marie; Guillaud, Laurent; Prola, Alexandre; Ferry, Arnaud; Aubin-Houzelstein, Geneviève; Demarquoy, Jean; Relaix, Frédéric; Piercy, Richard J; Blot, Stéphane; Kihara, Akio; Tiret, Laurent; Pilot-Storck, Fanny

    2015-10-01

    The reduced diameter of skeletal myofibres is a hallmark of several congenital myopathies, yet the underlying cellular and molecular mechanisms remain elusive. In this study, we investigate the role of HACD1/PTPLA, which is involved in the elongation of the very long chain fatty acids, in muscle fibre formation. In humans and dogs, HACD1 deficiency leads to a congenital myopathy with fibre size disproportion associated with a generalized muscle weakness. Through analysis of HACD1-deficient Labradors, Hacd1-knockout mice, and Hacd1-deficient myoblasts, we provide evidence that HACD1 promotes myoblast fusion during muscle development and regeneration. We further demonstrate that in normal differentiating myoblasts, expression of the catalytically active HACD1 isoform, which is encoded by a muscle-enriched splice variant, yields decreased lysophosphatidylcholine content, a potent inhibitor of myoblast fusion, and increased concentrations of ≥ C18 and monounsaturated fatty acids of phospholipids. These lipid modifications correlate with a reduction in plasma membrane rigidity. In conclusion, we propose that fusion impairment constitutes a novel, non-exclusive pathological mechanism operating in congenital myopathies and reveal that HACD1 is a key regulator of a lipid-dependent muscle fibre growth mechanism. PMID:26160855

  16. Effects of retroviral envelope-protein cleavage upon trafficking, incorporation, and membrane fusion

    SciTech Connect

    Apte, Swapna; Sanders, David Avram

    2010-09-15

    Retroviral envelope glycoproteins undergo proteolytic processing by cellular subtilisin-like proprotein convertases at a polybasic amino-acid site in order to produce the two functional subunits, SU and TM. Most previous studies have indicated that envelope-protein cleavage is required for rendering the protein competent for promoting membrane fusion and for virus infectivity. We have investigated the role of proteolytic processing of the Moloney murine leukemia virus envelope-protein through site-directed mutagenesis of the residues near the SU-TM cleavage site and have established that uncleaved glycoprotein is unable either to be incorporated into virus particles efficiently or to induce membrane fusion. Additionally, the results suggest that cleavage of the envelope protein plays an important role in intracellular trafficking of protein via the cellular secretory pathway. Based on our results it was concluded that a positively charged residue located at either P2 or P4 along with the arginine at P1 is essential for cleavage.

  17. Performance of a palladium membrane reactor using a Ni catalyst for fusion fuel impurities processing

    SciTech Connect

    Willms, R.S.; Wilhelm, R.; Okuno, K.

    1994-07-01

    The palladium membrane reactor (PNM) provides a means to recover hydrogen isotopes from impurities expected to be present in fusion reactor exhaust. This recovery is based on reactions such as water-gas shift and steam reforming for which conversion is equilibrium limited. By including a selectively permeable membrane such as Pd/Ag in the catalyst bed, hydrogen isotopes can be removed from the reacting environment, thus promoting the reaction to complete conversion. Such a device has been built and operated at the Tritium Systems Test Assembly (TSTA) at Los Alamos National Laboratory (LANL). For the reactions listed above, earlier study with this unit has shown that hydrogen single-pass recoveries approaching 100% can be achieved. It was also determined that a nickel catalyst is a feasible choice for use with a PMR appropriate for fusion fuel impurities processing. The purpose of this study was to systematically assess the performance of the PMR using a nickel catalyst over a range of temperatures, feed compositions and flowrates. Reactions which were studied are the water-gas shift reaction and steam reforming.

  18. Membrane Fusion Mediated Targeted Cytosolic Drug Delivery Through scFv Engineered Sendai Viral Envelopes.

    PubMed

    Kumar, M; Mani, P; Pratheesh, P; Chandra, S; Jeyakkodi, M; Chattopadhyay, P; Sarkar, D P; Sinha, S

    2015-01-01

    Antibody targeted cytoplasmic delivery of drugs is difficult to achieve as antigen-antibody interaction results in the payload being directed to the endosomal compartment. However, Sendai viral envelopes can bring about cytoplasmic delivery due to F-protein mediated membrane fusion. In this study we have generated and fused a recombinant scFv directed to the onco-fetal antigen, the Placental isozyme of Alkaline Phosphatase (PAP) with the trans-membrane and part of the cytoplasmic domain of the Sendai F protein (F(TMC)). Reconstituted virosomes, having both the fusion protein as well as the native F-protein were able to specifically bind and deliver drugs to PAP expressing cells. About 75% of the delivery was cytoplasmic in nature. Hence, this immuno-virosome, which is devoid of the comparatively more toxic HN protein, has the novel ability to combine specific antibody mediated targeting with cytoplasmic delivery. The scFv ensured specific binding to PAP expressing cells, without cross reacting with the other isozymes of alkaline phosphatase. The advantages of cytoplasmic delivery would include reduced degradation and lowered immunogenicity of the payload and carrier. The ubiquitous expression of PAP on a variety of cancers like seminoma, choriocarcinoma, cervical and breast cancers also suggests its potential usefulness in a number of malignancies.

  19. Reverse osmosis membrane composition, structure and performance modification by bisulphite, iron(III), bromide and chlorite exposure.

    PubMed

    Ferrer, O; Gibert, O; Cortina, J L

    2016-10-15

    Reverse osmosis (RO) membrane exposure to bisulphite, chlorite, bromide and iron(III) was assessed in terms of membrane composition, structure and performance. Membrane composition was determined by Rutherford backscattering spectrometry (RBS) and membrane performance was assessed by water and chloride permeation, using a modified version of the solution-diffusion model. Iron(III) dosage in presence of bisulphite led to an autooxidation of the latter, probably generating free radicals which damaged the membrane. It comprised a significant raise in chloride passage (chloride permeation coefficient increased 5.3-5.1 fold compared to the virgin membrane under the conditions studied) rapidly. No major differences in terms of water permeability and membrane composition were observed. Nevertheless, an increase in the size of the network pores, and a raise in the fraction of aggregate pores of the polyamide (PA) layer were identified, but no amide bond cleavage was observed. These structural changes were therefore, in accordance with the transport properties observed. PMID:27470468

  20. Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level.

    PubMed

    Otterstrom, Jason J; Brandenburg, Boerries; Koldijk, Martin H; Juraszek, Jarek; Tang, Chan; Mashaghi, Samaneh; Kwaks, Ted; Goudsmit, Jaap; Vogels, Ronald; Friesen, Robert H E; van Oijen, Antoine M

    2014-12-01

    The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action.

  1. Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

    PubMed Central

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A. Pedro; Starr, Trevor L.

    2015-01-01

    The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ΔPrm1 or Δlfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. PMID:25595444

  2. The Structure of Herpesvirus Fusion Glycoprotein B-Bilayer Complex Reveals the Protein-Membrane and Lateral Protein-Protein Interaction

    PubMed Central

    Maurer, Ulrike E.; Zeev-Ben-Mordehai, Tzviya; Pandurangan, Arun Prasad; Cairns, Tina M.; Hannah, Brian P.; Whitbeck, J. Charles; Eisenberg, Roselyn J.; Cohen, Gary H.; Topf, Maya; Huiskonen, Juha T.; Grünewald, Kay

    2013-01-01

    Summary Glycoprotein B (gB) is a key component of the complex herpesvirus fusion machinery. We studied membrane interaction of two gB ectodomain forms and present an electron cryotomography structure of the gB-bilayer complex. The two forms differed in presence or absence of the membrane proximal region (MPR) but showed an overall similar trimeric shape. The presence of the MPR impeded interaction with liposomes. In contrast, the MPR-lacking form interacted efficiently with liposomes. Lateral interaction resulted in coat formation on the membranes. The structure revealed that interaction of gB with membranes was mediated by the fusion loops and limited to the outer membrane leaflet. The observed intrinsic propensity of gB to cluster on membranes indicates an additional role of gB in driving the fusion process forward beyond the transient fusion pore opening and subsequently leading to fusion pore expansion. PMID:23850455

  3. Dissection of the Role of the Stable Signal Peptide of the Arenavirus Envelope Glycoprotein in Membrane Fusion

    PubMed Central

    Messina, Emily L.; York, Joanne

    2012-01-01

    The arenavirus envelope glycoprotein (GPC) retains a stable signal peptide (SSP) as an essential subunit in the mature complex. The 58-amino-acid residue SSP comprises two membrane-spanning hydrophobic regions separated by a short ectodomain loop that interacts with the G2 fusion subunit to promote pH-dependent membrane fusion. Small-molecule compounds that target this unique SSP-G2 interaction prevent arenavirus entry and infection. The interaction between SSP and G2 is sensitive to the phylogenetic distance between New World (Junín) and Old World (Lassa) arenaviruses. For example, heterotypic GPC complexes are unable to support virion entry. In this report, we demonstrate that the hybrid GPC complexes are properly assembled, proteolytically cleaved, and transported to the cell surface but are specifically defective in their membrane fusion activity. Chimeric SSP constructs reveal that this incompatibility is localized to the first transmembrane segment of SSP (TM1). Genetic changes in TM1 also affect sensitivity to small-molecule fusion inhibitors, generating resistance in some cases and inhibitor dependence in others. Our studies suggest that interactions of SSP TM1 with the transmembrane domain of G2 may be important for GPC-mediated membrane fusion and its inhibition. PMID:22438561

  4. Fusions involving protein kinase C and membrane-associated proteins in benign fibrous histiocytoma.

    PubMed

    Płaszczyca, Anna; Nilsson, Jenny; Magnusson, Linda; Brosjö, Otte; Larsson, Olle; Vult von Steyern, Fredrik; Domanski, Henryk A; Lilljebjörn, Henrik; Fioretos, Thoas; Tayebwa, Johnbosco; Mandahl, Nils; Nord, Karolin H; Mertens, Fredrik

    2014-08-01

    Benign fibrous histiocytoma (BFH) is a mesenchymal tumor that most often occurs in the skin (so-called dermatofibroma), but may also appear in soft tissues (so-called deep BFH) and in the skeleton (so-called non-ossifying fibroma). The origin of BFH is unknown, and it has been questioned whether it is a true neoplasm. Chromosome banding, fluorescence in situ hybridization, single nucleotide polymorphism arrays, RNA sequencing, RT-PCR and quantitative real-time PCR were used to search for recurrent somatic mutations in a series of BFH. BFHs were found to harbor recurrent fusions of genes encoding membrane-associated proteins (podoplanin, CD63 and LAMTOR1) with genes encoding protein kinase C (PKC) isoforms PRKCB and PRKCD. PKCs are serine-threonine kinases that through their many phosphorylation targets are implicated in a variety of cellular processes, as well as tumor development. When inactive, the amino-terminal, regulatory domain of PKCs suppresses the activity of their catalytic domain. Upon activation, which requires several steps, they typically translocate to cell membranes, where they interact with different signaling pathways. The detected PDPN-PRKCB, CD63-PRKCD and LAMTOR1-PRKCD gene fusions are all predicted to result in chimeric proteins consisting of the membrane-binding part of PDPN, CD63 or LAMTOR1 and the entire catalytic domain of the PKC. This novel pathogenetic mechanism should result in constitutive kinase activity at an ectopic location. The results show that BFH indeed is a true neoplasm, and that distorted PKC activity is essential for tumorigenesis. The findings also provide means to differentiate BFH from other skin and soft tissue tumors. This article is part of a Directed Issue entitled: Rare cancers.

  5. MORPHOLOGY OF GAMETE MEMBRANE FUSION AND OF SPERM ENTRY INTO OOCYTES OF THE SEA URCHIN

    PubMed Central

    Franklin, Luther E.

    1965-01-01

    Sea urchin gametes predominate in molecular studies of fertilization, yet relatively little is known of the subcellular aspects of sperm entry in this group. Accordingly, it seemed desirable to make a detailed examination of sperm entry phenomena in sea urchins with the electron microscope. Gametes of the sea urchins Arbacia punctulata and Lytechinus variegatus were used in this study. Samples of eggs containing 2 to 8 per cent oocytes were selected and fixed with osmium tetroxide in sea water at various intervals after insemination. Fixed specimens were embedded in Epon 812, sectioned, and examined with an electron microscope. An apical vesicle was observed at the anterior end of the acrosome. The presence of this structure, together with other observations, suggested that initiation of the acrosome reaction in sea urchin sperm involves dehiscence of the acrosomal region with the subsequent release of the acrosomal granule. Contact and initial fusion of gamete membranes was observed in mature eggs and oocytes and invariably involved the extended acrosomal tubule of the spermatozoon. Only one spermatozoon normally enters the mature egg. The probability of locating such a sperm in ultrathin sections is exceedingly low. Several sperm do normally enter oocytes. Consequently, observations of sperm entry were primarily restricted to the latter. The manner of sperm entry into oocytes did not resemble phagocytosis. Organelles of the spermatozoon were progressively divested of their plasma membrane as they entered the ground cytoplasm of the oocyte fertilization cone. Initiation of the acrosome reaction, contact and initial fusion of gamete membranes, and sperm entry into oocytes of sea urchins conform to the Hydroides-Saccoglossus pattern of early fertilization events as described by Colwin and Colwin (13). PMID:19866669

  6. Cdc42p and Rho1p are sequentially activated and mechanistically linked to vacuole membrane fusion

    SciTech Connect

    Logan, Michael R.; Jones, Lynden; Eitzen, Gary

    2010-03-26

    Small monomeric GTPases act as molecular switches, regulating many biological functions via activation of membrane localized signaling cascades. Activation of their switch function is controlled by GTP binding and hydrolysis. Two Rho GTPases, Cdc42p and Rho1p, are localized to the yeast vacuole where they regulate membrane fusion. Here, we define a method to directly examine vacuole membrane Cdc42p and Rho1p activation based on their affinity to probes derived from effectors. Cdc42p and Rho1p showed unique temporal activation which aligned with distinct subreactions of in vitro vacuole fusion. Cdc42p was rapidly activated in an ATP-independent manner while Rho1p activation was kinetically slower and required ATP. Inhibitors that are known to block vacuole membrane fusion were examined for their effect on Cdc42p and Rho1p activation. Rdi1p, which inhibits the dissociation of GDP from Rho proteins, blocked both Cdc42p and Rho1p activation. Ligands of PI(4,5)P{sub 2} specifically inhibited Rho1p activation while pre-incubation with U73122, which targets Plc1p function, increased Rho1p activation. These results define unique activation mechanisms for Cdc42p and Rho1p, which may be linked to the vacuole membrane fusion mechanism.

  7. Structure and membrane actions of a marine worm protein cytolysin, Cerebratulus toxin A-III.

    PubMed

    Kem, W R

    1994-02-28

    Four homologous Cerebratulus lacteus A toxins are the first and as yet only protein cytolysins to be isolated from an ancient phylum of marine worms, the nemertines. The most abundant and toxic variant, toxin A-III, has been sequenced and its mechanisms of action studied in the most detail. It consists of a single basic polypeptide chain of 95 amino acid residues cross-linked by three disulfide bonds, and possesses a predominantly alpha-helical secondary structure. The C-terminal third of the toxin sequence is postulated to be a helical 'hairpin' structure involved in pore formation. Toxin A-III permeabilizes a variety of cells as well as liposomes made from a variety of phospholipids; apparently large pores are formed, as large proteins are released almost as rapidly as small organic molecules and inorganic ions. At sublytic concentrations, the toxin also inhibits protein kinase C and endogenous voltage-gated cation selective (sodium, calcium) channels occurring in the nervous and cardiovascular systems. A curious observation, also reported for colicins and some other protein cytolysins, was the conservation of toxin secondary structure upon insertion into phospholipid liposomes, despite the strong likelihood that significant changes in tertiary structure occur to provide a hydrophobic surface for interaction with membrane lipids. Because of its small size and presumed single helical hairpin secondary structure, Cl toxin A-III is an excellent molecular subject for investigating protein insertion into biological membranes and mechanisms of pore formation.

  8. Live Salmonella recruits N-ethylmaleimide-sensitive fusion protein on phagosomal membrane and promotes fusion with early endosome.

    PubMed

    Mukherjee, K; Siddiqi, S A; Hashim, S; Raje, M; Basu, S K; Mukhopadhyay, A

    2000-02-21

    To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPgammaS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)-depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPgammaS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of alpha-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.

  9. A Structurally Unresolved Head Segment of Defined Length Favors Proper Measles Virus Hemagglutinin Tetramerization and Efficient Membrane Fusion Triggering

    PubMed Central

    Navaratnarajah, Chanakha K.; Rosemarie, Quincy

    2015-01-01

    ABSTRACT Paramyxoviruses include several insidious and ubiquitous pathogens of humans and animals, with measles virus (MeV) being a prominent one. The MeV membrane fusion apparatus consists of a receptor binding protein (hemagglutinin [H]) tetramer and a fusion (F) protein trimer. Four globular MeV H heads are connected to a tetrameric stalk through flexible linkers. We sought here to characterize the function of a 17-residue H-head segment proximal to the stalk that was unresolved in all five MeV H-head crystal or cocrystal structures. In particular, we assessed whether its primary sequence and length are critical for proper protein oligomerization and intracellular transport or for membrane fusion triggering. Extensive alanine substitutions had no effect on fusion triggering, suggesting that sequence identity is not critical for this function. Excessive shortening of this segment reduced or completely abrogated fusion trigger function, while length compensation restored it. We then characterized the mechanism of function loss. Mutated H proteins were efficiently transported to the cell surface, but certain alterations enhancing linker flexibility resulted in accumulation of high-molecular-weight H oligomers. Some oligomers had reduced fusion trigger capacity, while others retained this function. Thus, length and rigidity of the unresolved head segment favor proper H tetramerization and counteract interactions between subunits from different tetramers. The structurally unresolved H-head segment, together with the top of the stalk, may act as a leash to provide the right degree of freedom for the heads of individual tetramers to adopt a triggering-permissive conformation while avoiding improper contacts with heads of neighboring tetramers. IMPORTANCE Understanding the molecular mechanism of membrane fusion triggering may allow development of new antiviral strategies. The fusion apparatus of paramyxoviruses consists of a receptor binding tetramer and a fusion

  10. Fusion

    NASA Astrophysics Data System (ADS)

    Herman, Robin

    1990-10-01

    The book abounds with fascinating anecdotes about fusion's rocky path: the spurious claim by Argentine dictator Juan Peron in 1951 that his country had built a working fusion reactor, the rush by the United States to drop secrecy and publicize its fusion work as a propaganda offensive after the Russian success with Sputnik; the fortune Penthouse magazine publisher Bob Guccione sank into an unconventional fusion device, the skepticism that met an assertion by two University of Utah chemists in 1989 that they had created "cold fusion" in a bottle. Aimed at a general audience, the book describes the scientific basis of controlled fusion--the fusing of atomic nuclei, under conditions hotter than the sun, to release energy. Using personal recollections of scientists involved, it traces the history of this little-known international race that began during the Cold War in secret laboratories in the United States, Great Britain and the Soviet Union, and evolved into an astonishingly open collaboration between East and West.

  11. Rho GTPases and the Downstream Effectors Actin-related Protein 2/3 (Arp2/3) Complex and Myosin II Induce Membrane Fusion at Self-contacts*

    PubMed Central

    Sumida, Grant M.; Yamada, Soichiro

    2015-01-01

    Actin regulation is required for membrane activities that drive cell adhesion and migration. The Rho GTPase family plays critical roles in actin and membrane dynamics; however, the roles of the Rho GTPase family are not limited to cell adhesion and migration. Using micron-sized obstacles to induce the formation of self-contacts in epithelial cells, we previously showed that self-adhesion is distinct from cell-to-cell adhesion in that self-contacts are eliminated by membrane fusion. In the current study, we identified Rho GTPases, RhoA, Rac1, and Cdc42, as potential upstream regulators of membrane fusion. The RhoA downstream effector myosin II is required for fusion as the expression of mutant myosin light chain reduced membrane fusion. Furthermore, an inhibitor of the Arp2/3 complex, a downstream effector of Rac1 and Cdc42, also reduced self-contact-induced membrane fusion. At self-contacts, while the concentration of E-cadherin diminished, the intensity of GFP-tagged Arp3 rapidly fluctuated then decreased and stabilized after membrane fusion. Taken together, these data suggest that the Arp2/3 complex-mediated actin polymerization brings two opposing membranes into close apposition by possibly excluding E-cadherin from contact sites, thus promoting membrane fusion at self-contacts. PMID:25527498

  12. N-(3-Cyanophenyl)-2-phenylacetamide, an effective inhibitor of morbillivirus-induced membrane fusion with low cytotoxicity.

    PubMed

    Singethan, K; Hiltensperger, G; Kendl, S; Wohlfahrt, J; Plattet, P; Holzgrabe, U; Schneider-Schaulies, J

    2010-11-01

    Based on the structural similarity of viral fusion proteins within the family Paramyxoviridae, we tested recently described and newly synthesized acetanilide derivatives for their capacity to inhibit measles virus (MV)-, canine distemper virus (CDV)- and Nipah virus (NiV)-induced membrane fusion. We found that N-(3-cyanophenyl)-2-phenylacetamide (compound 1) has a high capacity to inhibit MV- and CDV-induced (IC(50) μM), but not NiV-induced, membrane fusion. This compound is of outstanding interest because it can be easily synthesized and its cytotoxicity is low [50 % cytotoxic concentration (CC(50)) ≥ 300 μM], leading to a CC(50)/IC(50) ratio of approximately 100. In addition, primary human peripheral blood lymphocytes and primary dog brain cell cultures (DBC) also tolerate high concentrations of compound 1. Infection of human PBMC with recombinant wild-type MV is inhibited by an IC(50) of approximately 20 μM. The cell-to-cell spread of recombinant wild-type CDV in persistently infected DBC can be nearly completely inhibited by compound 1 at 50 μM, indicating that the virus spread between brain cells is dependent on the activity of the viral fusion protein. Our findings demonstrate that this compound is a most applicable inhibitor of morbillivirus-induced membrane fusion in tissue culture experiments including highly sensitive primary cells. PMID:20685931

  13. Cr(III) exerts stronger structural effects than Cr(VI) on the human erythrocyte membrane and molecular models.

    PubMed

    Suwalsky, M; Castro, R; Villena, F; Sotomayor, C P

    2008-04-01

    Chromium exists in many oxidation states, of which only the hexavalent Cr(VI) and the trivalent Cr(III) ions are stable under environmental conditions. It is generally reported that Cr(VI) is highly toxic while Cr(III) is relatively innocuous, although others have reported just the opposite. On the other hand, despite the many studies on chromium toxicity, and particularly after the knowledge that Cr(VI) anions readily enter the erythrocytes where they are reduced to Cr(III), there are practically no reports on the structural effects induced by chromium compounds on the erythrocyte membrane. With the aim to better understand the molecular mechanisms of the interaction of Cr(III) and Cr(VI) with cell membranes, CrCl(3), and K(2)CrO(4) were incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of the erythrocyte membrane. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylcholine (DMPE), phospholipid classes present in the outer and inner monolayers of the erythrocyte membrane, respectively. The capacity of Cr(III) and Cr(VI) to perturb the bilayer structures of DMPC and DMPE was evaluated by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed with scanning electron microscopy (SEM). In all these systems, it was found that Cr(III) induced considerably higher structural perturbations than Cr(VI). PMID:18234343

  14. Early Events in Chikungunya Virus Infection—From Virus Cell Binding to Membrane Fusion

    PubMed Central

    van Duijl-Richter, Mareike K. S.; Hoornweg, Tabitha E.; Rodenhuis-Zybert, Izabela A.; Smit, Jolanda M.

    2015-01-01

    Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research. PMID:26198242

  15. Early Events in Chikungunya Virus Infection-From Virus Cell Binding to Membrane Fusion.

    PubMed

    van Duijl-Richter, Mareike K S; Hoornweg, Tabitha E; Rodenhuis-Zybert, Izabela A; Smit, Jolanda M

    2015-07-07

    Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research.

  16. Minor differences in the molecular machinery mediating regulated membrane fusion has major impact on metabolic health.

    PubMed

    Valladolid-Acebes, Ismael; Daraio, Teresa; Brismar, Kerstin; Hökfelt, Tomas; Bark, Christina

    2016-01-01

    The exocytosis of signaling molecules from neuronal, neuroendocrine and endocrine cells is regulated by membrane fusion involving SNAP-25 and associated SNARE proteins. The importance of this process for metabolic control recently became evident by studies of mouse mutants genetically engineered to only express one of 2 closely related, alternatively-spliced variants of SNAP-25. The results showed that even minor differences in the function of proteins regulating exocytosis are sufficient to provoke metabolic disease, including hyperglycaemia, liver steatosis, adipocyte hypertrophy and obesity. Thus, an imbalance in the dynamics of hormonal and/or neurotransmitter release can cause obesity and type 2 diabetes. This recent discovery highlights the fact that metabolic health requires a perfectly operating interplay between the SNARE protein machinery in excitable cells and the organs responding to these messengers. PMID:27617177

  17. Control systems for membrane fusion in the ancestral eukaryote; evolution of tethering complexes and SM proteins

    PubMed Central

    Koumandou, V Lila; Dacks, Joel B; Coulson, Richard MR; Field, Mark C

    2007-01-01

    Background In membrane trafficking, the mechanisms ensuring vesicle fusion specificity remain to be fully elucidated. Early models proposed that specificity was encoded entirely by SNARE proteins; more recent models include contributions from Rab proteins, Syntaxin-binding (SM) proteins and tethering factors. Most information on membrane trafficking derives from an evolutionarily narrow sampling of model organisms. However, considering factors from a wider diversity of eukaryotes can provide both functional information on core systems and insight into the evolutionary history of the trafficking machinery. For example, the major Qa/syntaxin SNARE families are present in most eukaryotic genomes and likely each evolved via gene duplication from a single ancestral syntaxin before the existing eukaryotic groups diversified. This pattern is also likely for Rabs and various other components of the membrane trafficking machinery. Results We performed comparative genomic and phylogenetic analyses, when relevant, on the SM proteins and components of the tethering complexes, both thought to contribute to vesicle fusion specificity. Despite evidence suggestive of secondary losses amongst many lineages, the tethering complexes are well represented across the eukaryotes, suggesting an origin predating the radiation of eukaryotic lineages. Further, whilst we detect distant sequence relations between GARP, COG, exocyst and DSL1 components, these similarities most likely reflect convergent evolution of similar secondary structural elements. No similarity is found between the TRAPP and HOPS complexes and the other tethering factors. Overall, our data favour independent origins for the various tethering complexes. The taxa examined possess at least one homologue of each of the four SM protein families; since the four monophyletic families each encompass a wide diversity of eukaryotes, the SM protein families very likely evolved before the last common eukaryotic ancestor (LCEA

  18. Polymeric membrane electrodes with high nitrite selectivity based on rhodium(III) porphyrins and salophens as ionophores.

    PubMed

    Pietrzak, Mariusz; Meyerhoff, Mark E

    2009-05-01

    Several porphyrin and salophen complexes with Rh(III) are examined as ionophores to prepare nitrite selective polymeric membrane electrodes. All ionophores tested exhibit preferred selectivity toward nitrite anion. Enhanced potentiometric nitrite selectivity is observed in the presence of either lipophilic anionic as well as cationic sites within the membranes, suggesting that the ionophores can function via either a charged or a neutral carrier response mechanism. Among a range of complexes and membrane formulations examined, optimal nitrite selectivity and reversible response down to 5 x 10(-6) M is achieved using Rh(III)-tetra(t-butylphenylporphyrin) as the ionophore in the presence of lipophilic cationic sites in plasticized poly(vinyl chloride) membrane. Response times are substantially longer than typical membrane electrodes apparently because of a slow nitrite ligation reaction with Rh(III); however, a significant improvement in dynamic EMF response can be realized by optimizing the membrane formulation and increasing the temperature. The selectivity observed with these membranes is greater than the best nitrite selective electrodes reported to date in the literature based on lipophilic Co(III)-corrin complexes, allowing the new nitrite electrodes to be utilized to determine the level of nitrite in meats with good correlation to the colorimetric Griess assay method.

  19. Fusion between perinuclear virions and the outer nuclear membrane requires the fusogenic activity of herpes simplex virus gB.

    PubMed

    Wright, Catherine C; Wisner, Todd W; Hannah, Brian P; Eisenberg, Roselyn J; Cohen, Gary H; Johnson, David C

    2009-11-01

    Herpesviruses cross nuclear membranes (NMs) in two steps, as follows: (i) capsids assemble and bud through the inner NM into the perinuclear space, producing enveloped virus particles, and (ii) the envelopes of these virus particles fuse with the outer NM. Two herpes simplex virus (HSV) glycoproteins, gB and gH (the latter, likely complexed as a heterodimer with gL), are necessary for the second step of this process. Mutants lacking both gB and gH accumulate in the perinuclear space or in herniations (membrane vesicles derived from the inner NM). Both gB and gH/gL are also known to act directly in fusing the virion envelope with host cell membranes during HSV entry into cells, i.e., both glycoproteins appear to function directly in different aspects of the membrane fusion process. We hypothesized that HSV gB and gH/gL also act directly in the membrane fusion that occurs during virus egress from the nucleus. Previous studies of the role of gB and gH/gL in nuclear egress involved HSV gB and gH null mutants that could potentially also possess gross defects in the virion envelope. Here, we produced recombinant HSV-expressing mutant forms of gB with single amino acid substitutions in the hydrophobic "fusion loops." These fusion loops are thought to play a direct role in membrane fusion by insertion into cellular membranes. HSV recombinants expressing gB with any one of four fusion loop mutations (W174R, W174Y, Y179K, and A261D) were unable to enter cells. Moreover, two of the mutants, W174Y and Y179K, displayed reduced abilities to mediate HSV cell-to-cell spread, and W174R and A261D exhibited no spread. All mutant viruses exhibited defects in nuclear egress, enveloped virions accumulated in herniations and in the perinuclear space, and fewer enveloped virions were detected on cell surfaces. These results support the hypothesis that gB functions directly to mediate the fusion between perinuclear virus particles and the outer NM.

  20. Membrane-Active Sequences within gp41 Membrane Proximal External Region (MPER) Modulate MPER-Containing Peptidyl Fusion Inhibitor Activity and the Biosynthesis of HIV-1 Structural Proteins

    PubMed Central

    Zhang, Si Min; Jejcic, Alenka; Tam, James P.; Vahlne, Anders

    2015-01-01

    The membrane proximal external region (MPER) is a highly conserved membrane-active region located at the juxtamembrane positions within class I viral fusion glycoproteins and essential for membrane fusion events during viral entry. The MPER in the human immunodeficiency virus type I (HIV-1) envelope protein (Env) interacts with the lipid bilayers through a cluster of tryptophan (Trp) residues and a C-terminal cholesterol-interacting motif. The inclusion of the MPER N-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-HIV peptidyl fusion inhibitors T20 and T1249. As a type I transmembrane protein, Env also interacts with the cellular membranes during its biosynthesis and trafficking. Here we investigated the roles of MPER membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. We found that elimination of the membrane-active elements in MPER peptides, namely, penta Trp→alanine (Ala) substitutions and the disruption of the C-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped HIV-1. Furthermore, as compared to C-terminal dimerization, N-terminal dimerization of MPER peptides and N-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. The secondary structure study revealed that the penta-Trp→Ala substitutions also increased the helical content in the MPER sequence, which prompted us to study the biological relevance of such mutations in pre-fusion Env. We observed that Ala mutations of Trp664, Trp668 and Trp670 in MPER moderately lowered the intracellular and intraviral contents of Env while significantly elevating the content of another viral structural protein, p55/Gag and its derivative p24/capsid. The data suggest a role of the gp41 MPER in the membrane-reactive events during

  1. Effect of the mounting membrane on shape in inertial confinement fusion implosions

    SciTech Connect

    Nagel, S. R. Haan, S. W.; Rygg, J. R.; Barrios, M.; Benedetti, L. R.; Bradley, D. K.; Field, J. E.; Hammel, B. A.; Izumi, N.; Jones, O. S.; Khan, S. F.; Ma, T.; Pak, A. E.; Tommasini, R.; Town, R. P. J.

    2015-02-15

    The performance of Inertial Confinement Fusion targets relies on the symmetric implosion of highly compressed fuel. X-ray area-backlit imaging is used to assess in-flight low mode 2D asymmetries of the shell. These time-resolved images of the shell exhibit features that can be related to the lift-off position of the membranes used to hold the capsule within the hohlraum. Here, we describe a systematic study of this membrane or “tent” thickness and its impact on the measured low modes for in-flight and self-emission images. The low mode amplitudes of the shell in-flight shape (P{sub 2} and P{sub 4}) are weakly affected by the tent feature in time-resolved, backlit data. By contrast, time integrated self-emission images along the same axis exhibit a reversal in perceived P{sub 4} mode due to growth of a feature seeded by the tent, which can explain prior inconsistencies between the in-flight P{sub 4} and core P{sub 4}, leading to a reevaluation of optimum hohlraum length. Simulations with a tent-like feature normalized to match the feature seen in the backlit images predict a very large impact on the capsule performance from the tent feature.

  2. DNA release from lipoplexes by anionic lipids: correlation with lipid mesomorphism, interfacial curvature, and membrane fusion

    SciTech Connect

    Tarahovsky, Yury S.; Koynova, Rumiana; MacDonald, Robert C.

    2010-01-18

    DNA release from lipoplexes is an essential step during lipofection and is probably a result of charge neutralization by cellular anionic lipids. As a model system to test this possibility, fluorescence resonance energy transfer between DNA and lipid covalently labeled with Cy3 and BODIPY, respectively, was used to monitor the release of DNA from lipid surfaces induced by anionic liposomes. The separation of DNA from lipid measured this way was considerably slower and less complete than that estimated with noncovalently labeled DNA, and depends on the lipid composition of both lipoplexes and anionic liposomes. This result was confirmed by centrifugal separation of released DNA and lipid. X-ray diffraction revealed a clear correlation of the DNA release capacity of the anionic lipids with the interfacial curvature of the mesomorphic structures developed when the anionic and cationic liposomes were mixed. DNA release also correlated with the rate of fusion of anionic liposomes with lipoplexes. It is concluded that the tendency to fuse and the phase preference of the mixed lipid membranes are key factors for the rate and extent of DNA release. The approach presented emphasizes the importance of the lipid composition of both lipoplexes and target membranes and suggests optimal transfection may be obtained by tailoring lipoplex composition to the lipid composition of target cells.

  3. Direct targeting of membrane fusion by SNARE mimicry: Convergent evolution of Legionella effectors

    PubMed Central

    Shi, Xingqi; Halder, Partho; Yavuz, Halenur; Shuman, Howard A.

    2016-01-01

    Legionella pneumophila, the Gram-negative pathogen causing Legionnaires’ disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNARE mimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host’s disassembly machinery, thus effectively trapping VAMP4 in an inactive state. PMID:27436892

  4. Direct targeting of membrane fusion by SNARE mimicry: Convergent evolution of Legionella effectors.

    PubMed

    Shi, Xingqi; Halder, Partho; Yavuz, Halenur; Jahn, Reinhard; Shuman, Howard A

    2016-08-01

    Legionella pneumophila, the Gram-negative pathogen causing Legionnaires' disease, infects host cells by hijacking endocytic pathways and forming a Legionella-containing vacuole (LCV) in which the bacteria replicate. To promote LCV expansion and prevent lysosomal targeting, effector proteins are translocated into the host cell where they alter membrane traffic. Here we show that three of these effectors [LegC2 (Legionella eukaryotic-like gene C2)/YlfB (yeast lethal factor B), LegC3, and LegC7/YlfA] functionally mimic glutamine (Q)-SNARE proteins. In infected cells, the three proteins selectively form complexes with the endosomal arginine (R)-SNARE vesicle-associated membrane protein 4 (VAMP4). When reconstituted in proteoliposomes, these proteins avidly fuse with liposomes containing VAMP4, resulting in a stable complex with properties resembling canonical SNARE complexes. Intriguingly, however, the LegC/SNARE hybrid complex cannot be disassembled by N-ethylmaleimide-sensitive factor. We conclude that LegCs use SNARE mimicry to divert VAMP4-containing vesicles for fusion with the LCV, thus promoting its expansion. In addition, the LegC/VAMP4 complex avoids the host's disassembly machinery, thus effectively trapping VAMP4 in an inactive state. PMID:27436892

  5. Cholesterol-Dependent Membrane Fusion Induced by the gp41 Membrane-Proximal External Region–Transmembrane Domain Connection Suggests a Mechanism for Broad HIV-1 Neutralization

    PubMed Central

    Apellániz, Beatriz; Rujas, Edurne; Carravilla, Pablo; Requejo-Isidro, José; Huarte, Nerea

    2014-01-01

    ABSTRACT The HIV-1 glycoprotein 41 promotes fusion of the viral membrane with that of the target cell. Structural, biochemical, and biophysical studies suggest that its membrane-proximal external region (MPER) may interact with the HIV-1 membrane and induce its disruption and/or deformation during the process. However, the high cholesterol content of the envelope (ca. 40 to 50 mol%) imparts high rigidity, thereby acting against lipid bilayer restructuring. Here, based on the outcome of vesicle stability assays, all-atom molecular dynamics simulations, and atomic force microscopy observations, we propose that the conserved sequence connecting the MPER with the N-terminal residues of the transmembrane domain (TMD) is involved in HIV-1 fusion. This junction would function by inducing phospholipid protrusion and acyl-chain splay in the cholesterol-enriched rigid envelope. Supporting the functional relevance of such a mechanism, membrane fusion was inhibited by the broadly neutralizing 4E10 antibody but not by a nonneutralizing variant with the CDR-H3 loop deleted. We conclude that the MPER-TMD junction embodies an envelope-disrupting C-terminal fusion peptide that can be targeted by broadly neutralizing antibodies. IMPORTANCE Fusion of the cholesterol-enriched viral envelope with the cell membrane marks the beginning of the infectious HIV-1 replicative cycle. Consequently, the Env glycoprotein-mediated fusion function constitutes an important clinical target for inhibitors and preventive vaccines. Antibodies 4E10 and 10E8 bind to one Env vulnerability site located at the gp41 membrane-proximal external region (MPER)–transmembrane domain (TMD) junction and block infection. These antibodies display broad viral neutralization, which underscores the conservation and functionality of the MPER-TMD region. In this work, we combined biochemical assays with molecular dynamics simulations and microscopy observations to characterize the unprecedented fusogenic activity of the

  6. Chromium(III) selective membrane sensors based on Schiff bases as chelating ionophores.

    PubMed

    Singh, A K; Gupta, V K; Gupta, Barkha

    2007-02-28

    The two chromium chelates of Schiff bases, N-(acetoacetanilide)-1,2-diaminoethane (L(1)) and N,N'-bis(acetoacetanilide)-triethylenetetraammine (L(2)), have been synthesized and explored as neutral ionophores for preparing poly(vinylchloride) (PVC) based membrane sensors selective to Cr(III). The addition of lipophilic anion excluder (NaTPB) and various plasticizers viz. o-Nitrophenyloctyl ether (o-NPOE), dioctylpthalate (DOP), dibutylphthalate (DBP), tris(2-ethylhexyl)phosphate (TEHP), and benzyl acetate (BA) have found to improve the performance of the sensors. The best performance was obtained for the membrane sensor having a composition of L(1):PVC:DBP:NaTPB in the ratio 5:150:250:3 (w/w). The sensor exhibits Nernstian response in the concentration range 8.9 x 10(-8) to 1.0 x 10(-1) M Cr(3+) with limit of detection 5.6 x 10(-8) M. The proposed sensor manifest advantages of relatively fast response (10s) and good selectivity over some alkali, alkaline earth, transition and heavy metal ions. The selectivity behavior of the proposed electrode revealed a considerable improvement as compared to the best previously PVC-membrane electrode for chromium(III) ion. The potentiometric response of the proposed sensor was independent of pH of the test solution in the range of 2.0-7.0. The sensor has found to work satisfactorily in partially non-aqueous media up to 20% (v/v) content of methanol, ethanol and acetonitrile and could be used for a period of 3 months. The proposed electrode was used as an indicator electrode in potentiometric titration of chromium ion with EDTA and in direct determination in different water and food samples.

  7. CNS myelin sheath is stochastically built by homotypic fusion of myelin membranes within the bounds of an oligodendrocyte process.

    PubMed

    Szuchet, Sara; Nielsen, Lauren L; Domowicz, Miriam S; Austin, Jotham R; Arvanitis, Dimitrios L

    2015-04-01

    Myelin - the multilayer membrane that envelops axons - is a facilitator of rapid nerve conduction. Oligodendrocytes form CNS myelin; the prevailing hypothesis being that they do it by extending a process that circumnavigates the axon. It is pertinent to ask how myelin is built because oligodendrocyte plasma membrane and myelin are compositionally different. To this end, we examined oligodendrocyte cultures and embryonic avian optic nerves by electron microscopy, immuno-electron microscopy and three-dimensional electron tomography. The results support three novel concepts. Myelin membranes are synthesized as tubules and packaged into "myelinophore organelles" in the oligodendrocyte perikaryon. Myelin membranes are matured in and transported by myelinophore organelles within an oligodendrocyte process. The myelin sheath is generated by myelin membrane fusion inside an oligodendrocyte process. These findings abrogate the dogma of myelin resulting from a wrapping motion of an oligodendrocyte process and open up new avenues in the quest for understanding myelination in health and disease.

  8. Structure of a bacterial type III secretion system in contact with a host membrane in situ

    PubMed Central

    Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R.; Hayward, Richard D.

    2015-01-01

    Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform–ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking ‘pump-action' conformational changes that underpin effector injection. PMID:26656452

  9. Structure of a bacterial type III secretion system in contact with a host membrane in situ

    NASA Astrophysics Data System (ADS)

    Nans, Andrea; Kudryashev, Mikhail; Saibil, Helen R.; Hayward, Richard D.

    2015-12-01

    Many bacterial pathogens of animals and plants use a conserved type III secretion system (T3SS) to inject virulence effector proteins directly into eukaryotic cells to subvert host functions. Contact with host membranes is critical for T3SS activation, yet little is known about T3SS architecture in this state or the conformational changes that drive effector translocation. Here we use cryo-electron tomography and sub-tomogram averaging to derive the intact structure of the primordial Chlamydia trachomatis T3SS in the presence and absence of host membrane contact. Comparison of the averaged structures demonstrates a marked compaction of the basal body (4 nm) occurs when the needle tip contacts the host cell membrane. This compaction is coupled to a stabilization of the cytosolic sorting platform-ATPase. Our findings reveal the first structure of a bacterial T3SS from a major human pathogen engaged with a eukaryotic host, and reveal striking `pump-action' conformational changes that underpin effector injection.

  10. Crystal Structure of Dengue Type 1 Envelope Protein in the Postfusion Conformation and its Implication for Receptor Binding, Membrane Fusion and Antibody Recognition

    SciTech Connect

    Nayak, V.; Dessau, M; Kucera, K; Anthony, K; Ledizet, M; Modis, Y

    2009-01-01

    Dengue virus relies on a conformational change in its envelope protein, E, to fuse the viral lipid membrane with the endosomal membrane and thereby deliver the viral genome into the cytosol. We have determined the crystal structure of a soluble fragment E (sE) of dengue virus type 1 (DEN-1). The protein is in the postfusion conformation even though it was not exposed to a lipid membrane or detergent. At the domain I-domain III interface, 4 polar residues form a tight cluster that is absent in other flaviviral postfusion structures. Two of these residues, His-282 and His-317, are conserved in flaviviruses and are part of the pH sensor that triggers the fusogenic conformational change in E, at the reduced pH of the endosome. In the fusion loop, Phe-108 adopts a distinct conformation, forming additional trimer contacts and filling the bowl-shaped concavity observed at the tip of the DEN-2 sE trimer.

  11. SNARE-mediated rapid lysosome fusion in membrane raft clustering and dysfunction of bovine coronary arterial endothelium

    PubMed Central

    Han, Wei-Qing; Xia, Min; Zhang, Chun; Zhang, Fan; Xu, Ming; Li, Ning-Jun

    2011-01-01

    The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arteries. Direct lysosome fusion monitoring by N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide (FM1-43) quenching demonstrated that the inhibition of VAMP-2 with tetanus toxin or specific small interfering ribonucleic acid (siRNA) almost completely blocked lysosome fusion to plasma membrane induced by Fas ligand (FasL), a well-known MR clustering stimulator. The involvement of SNAREs was further confirmed by an increased interaction of VAMP-2 with a target-SNARE protein syntaxin-4 after FasL stimulation in coimmunoprecipitation analysis. Also, the inhibition of VAMP-2 with tetanus toxin or VAMP-2 siRNA abolished FasL-induced MR clustering, its colocalization with a NADPH oxidase unit gp91phox, and increased superoxide production. Finally, FasL-induced impairment of endothelium-dependent vasodilation was reversed by the treatment of bovine coronary arteries with tetanus toxin or VAMP-2 siRNA. VAMP-2 is critical to lysosome fusion in MR clustering, and this VAMP-2-mediated lysosome-MR signalosomes contribute to redox regulation of coronary endothelial function. PMID:21926345

  12. Alkylation by secondary alcohols III: Fusion of medicinal sulfanilamides with benzhydrol.

    PubMed

    Moskalyk, R E; Malicky, J L

    1975-02-01

    The fusion of certain sulfanilamides with benzhydrol in the presence of anhydrous zinc chloride affords several different products, depending primarily on the temperature at which the reaction is carried out. With sulfanilamide itself, three different products were isolated at 100, 160, and 180 degrees. A sequence of steps is suggested to account for the three products, one of which involves an intramolecular rearrangement of a benzhydryl moiety. The fusion of benzhydrol with p-toludine gives 2,6-dibenzhydrylaniline and not the N,N-dibenzhydryl derivative as previously reported.

  13. The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms.

    PubMed

    Siegel, D P; Epand, R M

    1997-12-01

    We studied the mechanism of the lamellar-to-inverted hexagonal (L alpha/H[II]) phase transition, using time-resolved cryotransmission electron microscopy (TRC-TEM), 31P-NMR, and differential scanning calorimetry. The transition was initiated in dispersions of large unilamellar vesicles of dipalmitoleoyl phosphatidylethanolamine (DiPoPE). We present evidence that the transition proceeds in three steps. First, many small connections form between apposed membranes. Second, the connections aggregate within the planes of the bilayers, forming arrays with hexagonal order in some projections. Third, these quasihexagonal structures elongate into small domains of H(II) phase, acquiring lipid molecules by diffusion from contiguous bilayers. A previously proposed membrane fusion mechanism rationalizes these results. The modified stalk theory predicts that the L alpha/H(II) phase transition involves some of the same intermediate structures as membrane fusion. The small interbilayer connections observed via TRC-TEM are compatible with the structure of a critical intermediate in the modified stalk mechanism: the trans monolayer contact (TMC). The theory predicts that 1) TMCs should form starting at tens of degrees below TH; 2) when TMCs become sufficiently numerous, they should aggregate into transient arrays like the quasihexagonal arrays observed here by TRC-TEM; and 3) these quasihexagonal arrays can then elongate directly into H(II) phase domains. These predictions rationalize the principal features of our data, which are incompatible with the other transition mechanisms proposed to date. Thus these results support the modified stalk mechanism for both membrane fusion and the L alpha/H(II) phase transition. We also discuss some implications of the modified stalk theory for fusion in protein-containing systems. Specifically, we point out that recent data on the effects of hydrophobic peptides and viral fusion peptides on lipid phase behavior are consistent with an effect of

  14. The mechanism of lamellar-to-inverted hexagonal phase transitions in phosphatidylethanolamine: implications for membrane fusion mechanisms.

    PubMed Central

    Siegel, D P; Epand, R M

    1997-01-01

    We studied the mechanism of the lamellar-to-inverted hexagonal (L alpha/H[II]) phase transition, using time-resolved cryotransmission electron microscopy (TRC-TEM), 31P-NMR, and differential scanning calorimetry. The transition was initiated in dispersions of large unilamellar vesicles of dipalmitoleoyl phosphatidylethanolamine (DiPoPE). We present evidence that the transition proceeds in three steps. First, many small connections form between apposed membranes. Second, the connections aggregate within the planes of the bilayers, forming arrays with hexagonal order in some projections. Third, these quasihexagonal structures elongate into small domains of H(II) phase, acquiring lipid molecules by diffusion from contiguous bilayers. A previously proposed membrane fusion mechanism rationalizes these results. The modified stalk theory predicts that the L alpha/H(II) phase transition involves some of the same intermediate structures as membrane fusion. The small interbilayer connections observed via TRC-TEM are compatible with the structure of a critical intermediate in the modified stalk mechanism: the trans monolayer contact (TMC). The theory predicts that 1) TMCs should form starting at tens of degrees below TH; 2) when TMCs become sufficiently numerous, they should aggregate into transient arrays like the quasihexagonal arrays observed here by TRC-TEM; and 3) these quasihexagonal arrays can then elongate directly into H(II) phase domains. These predictions rationalize the principal features of our data, which are incompatible with the other transition mechanisms proposed to date. Thus these results support the modified stalk mechanism for both membrane fusion and the L alpha/H(II) phase transition. We also discuss some implications of the modified stalk theory for fusion in protein-containing systems. Specifically, we point out that recent data on the effects of hydrophobic peptides and viral fusion peptides on lipid phase behavior are consistent with an effect of

  15. Polymeric Membrane Electrodes with High Nitrite Selectivity Based on Rhodium(III) Porphyrins and Salophens as Ionophores

    PubMed Central

    Pietrzak, Mariusz; Meyerhoff, Mark E.

    2009-01-01

    Several porphyrin and salophen complexes with Rh(III) are examined as ionophores to prepare nitrite selective polymeric membrane electrodes. All ionophores tested exhibit preferred selectivity towards nitrite anion. Enhanced potentiometric nitrite selectivity is observed in the presence of either lipophilic anionic as well as cationic sites within the membranes, suggesting that the ionophores can function via either a charged or neutral carrier response mechanism. Among a range of complexes and membrane formulations examined, optimal nitrite selectivity and reversible response down to 5 × 10−6 M is achieved using Rh(III)-tetra(t-butyl-phenylporphyrin) as the ionophore in the presence of lipophilic cationic sites in plasticized PVC membrane. Response times are substantially longer than typical membrane electrodes apparently due to slow nitrite ligation reaction however, a significant improvement in dynamic EMF response can be realized by optimizing the membrane formulation and increasing temperature. The selecitivity observed with these membranes is greater than the best nitrite selective electrodes reported to the date in the literature based on lipophilic Co(III)-corrin complexes, allowing the new nitrite electrodes to be utilized to determine the level of nitrite in meats with good correlation to the colorimetric Griess assay method. PMID:19402723

  16. Study of Cobalt(III) Corrole as the Neutral Ionophore for Nitrite and Nitrate Detection via Polymeric Membrane Electrodes

    PubMed Central

    Yang, Si; Meyerhoff, Mark E.

    2014-01-01

    Cobalt(III) 5, 10, 15-tris(4-tert-butylphenyl) corrole was synthesized and incorporated into plasticized poly(vinyl chloride) membranes and studied as a neutral carrier ionophore via potentiometry. This cobalt(III) complex has binding affinity to nitrite, and the resulting membrane electrode yields reversible and Nernstian response toward nitrite. Enhanced nitrite selectivity is observed over other anions, including lipophilic anions such as thiocyanate and perchlorate when an appropriate amount of lipophilic cationic sites are added to the membrane phase. Detection limit to nitrite is ca. 5 µM. Using tributylphosphate as the plasticizer with the cobalt(III) corrole species yields electrodes with enhanced nitrate selectivity. PMID:25422577

  17. The role of blood cell membrane lipids on the mode of action of HIV-1 fusion inhibitor sifuvirtide

    SciTech Connect

    Matos, Pedro M.; Freitas, Teresa; Castanho, Miguel A.R.B.; Santos, Nuno C.

    2010-12-17

    Research highlights: {yields} Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. {yields} Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. {yields} This peptide-membrane interaction may direct the drug towards raft-like membrane domains where the receptors used by HIV are located, facilitating its inhibitory action. -- Abstract: Sifuvirtide is a gp41 based peptide that inhibits HIV-1 fusion with the host cells and is currently under clinical trials. Previous studies showed that sifuvirtide partitions preferably to saturated phosphatidylcholine lipid membranes, instead of fluid-phase lipid vesicles. We extended the study to the interaction of the peptide with circulating blood cells, by using the dipole potential sensitive probe di-8-ANEPPS. Sifuvirtide decreased the dipole potential of erythrocyte and lymphocyte membranes in a concentration dependent manner, demonstrating its interaction. Also, the lipid selectivity of the peptide towards more rigid phosphatidylcholines was confirmed based on the dipole potential variations. Overall, the interaction of the peptide with the cell membranes is a contribution of different lipid preferences that presumably directs the peptide towards raft-like domains where the receptors are located, facilitating the reach of the peptide to its molecular target, the gp41 in its pre-fusion conformation.

  18. Effects of transmembrane potential and pH gradient on the cytochrome c-promoted fusion of mitochondrial mimetic membranes.

    PubMed

    Kawai, Cintia; Pessoto, Felipe S; Graves, Catharine V; Carmona-Ribeiro, Ana Maria; Nantes, Iseli L

    2013-08-01

    The present study investigated the effects of ΔΨ and ΔpH (pH gradient) on the interaction of cytochrome c with a mitochondrial mimetic membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) leading to vesicle fusion. ΔpH generated by lowered bulk pH (pH(out)) of PCPECL liposomes, with an internal pH (pH(in)) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pK(a) ~ 6.9). Conversely, ΔpH generated by enhanced pH(in) of PCPECL at a pH(out) of 6.0 favored the fusion of vesicles with a linear profile. We did not observe a significant amount of liposome fusion when ΔpH was generated by lowered pH(in) at a pH(out) of 8.0. At bulk acidic pH, ΔΨ generated by Na⁺ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pH(out), the presence of ΔpH and ΔΨ did not affect cytochrome c binding affinity measured by pyrene quenching. Therefore, cytochrome c-mediated PC/PE/CL vesicle fusion is dependent of ionization of the protein site L (acidic pH) and the presence of transmembrane potential. The effect of transmembrane potential is probably related to the generation of defects on the lipid bilayer. These results are consistent with previous reports showing that cytochrome c release prior to the dissipation of the ΔΨ(M) blocks inner mitochondrial membrane fusion during apoptosis.

  19. Kinetically coupled folding of a single HIV-1 glycoprotein 41 complex in viral membrane fusion and inhibition

    PubMed Central

    Jiao, Junyi; Rebane, Aleksander A.; Ma, Lu; Gao, Ying; Zhang, Yongli

    2015-01-01

    HIV-1 glycoprotein 41 (gp41) mediates viral entry into host cells by coupling its folding energy to membrane fusion. Gp41 folding is blocked by fusion inhibitors, including the commercial drug T20, to treat HIV/AIDS. However, gp41 folding intermediates, energy, and kinetics are poorly understood. Here, we identified the folding intermediates of a single gp41 trimer-of-hairpins and measured their associated energy and kinetics using high-resolution optical tweezers. We found that folding of gp41 hairpins was energetically independent but kinetically coupled: Each hairpin contributed a folding energy of ∼−23 kBT, but folding of one hairpin successively accelerated the folding rate of the next one by ∼20-fold. Membrane-mimicking micelles slowed down gp41 folding and reduced the stability of the six-helix bundle. However, the stability was restored by cooperative folding of the membrane-proximal external region. Surprisingly, T20 strongly inhibited gp41 folding by actively displacing the C-terminal hairpin strand in a force-dependent manner. The inhibition was abolished by a T20-resistant gp41 mutation. The energetics and kinetics of gp41 folding established by us provides a basis to understand viral membrane fusion, infection, and therapeutic intervention. PMID:26038562

  20. Physics of laser fusion. Volume III. High-power pulsed lasers

    SciTech Connect

    Holzrichter, J.F.; Eimerl, D.; George, E.V.; Trenholme, J.B.; Simmons, W.W.; Hunt, J.T.

    1982-09-01

    High-power pulsed lasers can deliver sufficient energy on inertial-confinement fusion (ICF) time scales (0.1 to 10 ns) to heat and compress deuterium-tritium fuel to fusion-reaction conditions. Several laser systems have been examined, including Nd:glass, CO/sub 2/, KrF, and I/sub 2/, for their ICF applicability. A great deal of developmental effort has been applied to the Nd:glass laser and the CO/sub 2/ gas laser systems; these systems now deliver > 10/sup 4/ J and 20 x 10/sup 12/ W to ICF targets. We are constructing the Nova Nd:glass laser at LLNL to provide > 100 kJ and > 100 x 10/sup 12/ W of 1-..mu..m radiation for fusion experimentation in the mid-1980s. For ICF target gain > 100 times the laser input, we expect that the laser driver must deliver approx. 3 to 5 MJ of energy on a time scale of 10 to 20 ns. In this paper we review the technological status of fusion-laser systems and outline approaches to constructing high-power pulsed laser drivers.

  1. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains.

    PubMed

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxPhi domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1(NL4.3) compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

  2. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

    SciTech Connect

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the Yxx{phi} domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1{sub NL4.3} compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and

  3. Full-length trimeric influenza virus hemagglutinin II membrane fusion protein and shorter constructs lacking the fusion peptide or transmembrane domain: Hyperthermostability of the full-length protein and the soluble ectodomain and fusion peptide make significant contributions to fusion of membrane vesicles.

    PubMed

    Ratnayake, Punsisi U; Prabodha Ekanayaka, E A; Komanduru, Sweta S; Weliky, David P

    2016-01-01

    Influenza virus is a class I enveloped virus which is initially endocytosed into a host respiratory epithelial cell. Subsequent reduction of the pH to the 5-6 range triggers a structural change of the viral hemagglutinin II (HA2) protein, fusion of the viral and endosomal membranes, and release of the viral nucleocapsid into the cytoplasm. HA2 contains fusion peptide (FP), soluble ectodomain (SE), transmembrane (TM), and intraviral domains with respective lengths of ∼ 25, ∼ 160, ∼ 25, and ∼ 10 residues. The present work provides a straightforward protocol for producing and purifying mg quantities of full-length HA2 from expression in bacteria. Biophysical and structural comparisons are made between full-length HA2 and shorter constructs including SHA2 ≡ SE, FHA2 ≡ FP+SE, and SHA2-TM ≡ SE+TM constructs. The constructs are helical in detergent at pH 7.4 and the dominant trimer species. The proteins are highly thermostable in decylmaltoside detergent with Tm>90 °C for HA2 with stabilization provided by the SE, FP, and TM domains. The proteins are likely in a trimer-of-hairpins structure, the final protein state during fusion. All constructs induce fusion of negatively-charged vesicles at pH 5.0 with much less fusion at pH 7.4. Attractive protein/vesicle electrostatics play a role in fusion, as the proteins are positively-charged at pH 5.0 and negatively-charged at pH 7.4 and the pH-dependence of fusion is reversed for positively-charged vesicles. Comparison of fusion between constructs supports significant contributions to fusion from the SE and the FP with little effect from the TM.

  4. Different activities of the reovirus FAST proteins and influenza hemagglutinin in cell-cell fusion assays and in response to membrane curvature agents

    SciTech Connect

    Clancy, Eileen K.; Barry, Chris; Ciechonska, Marta; Duncan, Roy

    2010-02-05

    The reovirus fusion-associated small transmembrane (FAST) proteins evolved to induce cell-cell, rather than virus-cell, membrane fusion. It is unclear whether the FAST protein fusion reaction proceeds in the same manner as the enveloped virus fusion proteins. We now show that fluorescence-based cell-cell and cell-RBC hemifusion assays are unsuited for detecting lipid mixing in the absence of content mixing during FAST protein-mediated membrane fusion. Furthermore, membrane curvature agents that inhibit hemifusion or promote pore formation mediated by influenza hemagglutinin had no effect on p14-induced cell-cell fusion, even under conditions of limiting p14 concentrations. Standard assays used to detect fusion intermediates induced by enveloped virus fusion proteins are therefore not applicable to the FAST proteins. These results suggest the possibility that the nature of the fusion intermediates or the mechanisms used to transit through the various stages of the fusion reaction may differ between these distinct classes of viral fusogens.

  5. Membrane potential and endocytic activity control disintegration of cell-cell adhesion and cell fusion in vinculin-injected MDBK cells.

    PubMed

    Palovuori, Riitta; Myrsky, Essi; Eskelinen, Sinikka

    2004-09-01

    Cell fusion occurs during fertilization and in the formation of organs such as muscles, placenta, and bones. We have developed an experimental model for epithelial cell fusion which permits analysis of the processes during junction disintegration and formation of polykaryons (Palovuori and Eskelinen [2000] Eur. J. Cell. Biol. 79: 961-974). In the present work, we analyzed the process in detail. Cell fusion was achieved by microinjecting into the cytoplasm of kidney epithelial Madin-Darby bovine kidney (MDBK) cells TAMRA-tagged vinculin, which incorporated into lateral membranes, focal adhesions and nucleus, and, prior fusion, induced internalization of actin, cadherin and plakoglobin to small clusters in cytoplasm. Injected vinculin was still visible at lateral membranes after removal of junctional proteins indicating that it was tightly associated and perturbed the cell-cell contact sites resulting in membrane fragmentation. Injection of active Rac together with vinculin induced accumulation of cadherin to the membranes, but did not affect vinculin-membrane association. However, it hampered cell fusion probably by supporting adherens junctions. In order to stop endocytosis, we lowered intracellular pH of vinculin-injected cells to 5.5 with the aid of nigericin in KCl buffer. In acidified cells, injected vinculin delineated lateral membranes as thick layers, cadherin remained in situ, and cell fusion was completely inhibited. Since this treatment also leads to cell depolarization, we checked the vinculin incorporation in a KCl solution containing nigericin at neutral pH. In these circumstances, both endogenous and injected vinculin delineated lateral membranes as very thin discontinuous layers, but still fusion was hampered most likely due to perturbation in the initial vinculin-membrane association. We suggest that vinculin might function as a sensor of the environment triggering cell fusion during development in circumstances where membrane potential and local

  6. A gene-fusion strategy for stoichiometric and co-localized expression of light-gated membrane proteins.

    PubMed

    Kleinlogel, Sonja; Terpitz, Ulrich; Legrum, Barbara; Gökbuget, Deniz; Boyden, Edward S; Bamann, Christian; Wood, Phillip G; Bamberg, Ernst

    2011-12-01

    The precise co-localization and stoichiometric expression of two different light-gated membrane proteins can vastly improve the physiological usefulness of optogenetics for the modulation of cell excitability with light. Here we present a gene-fusion strategy for the stable 1:1 expression of any two microbial rhodopsins in a single polypeptide chain. By joining the excitatory channelrhodopsin-2 with the inhibitory ion pumps halorhodopsin or bacteriorhodopsin, we demonstrate light-regulated quantitative bi-directional control of the membrane potential in HEK293 cells and neurons in vitro. We also present synergistic rhodopsin combinations of channelrhodopsin-2 with Volvox carteri channelrhodopsin-1 or slow channelrhodopsin-2 mutants, to achieve enhanced spectral or kinetic properties, respectively. Finally, we demonstrate the utility of our fusion strategy to determine ion-turnovers of as yet uncharacterized rhodopsins, exemplified for archaerhodopsin and CatCh, or to correct pump cycles, exemplified for halorhodopsin. PMID:22056675

  7. Membrane fusion intermediates and the effect of cholesterol: An in-house X-ray scattering study

    NASA Astrophysics Data System (ADS)

    Aeffner, S.; Reusch, T.; Weinhausen, B.; Salditt, T.

    2009-10-01

    We have developed an X-ray scattering setup which allows to study membrane fusion intermediates or other nonlamellar lipid mesophases by laboratory-scale X-ray sources alone, thus taking advantage of unrestricted beamtime compared to synchrotron sources. We report results of a study of pure lipid bilayers and phospholipid/cholesterol binary mixtures. Stalks, putative intermediate structures occurring during the membrane fusion process, can clearly be identified from reconstructed electron density maps. Phase diagrams of the lyotropic phase behavior of DOPC/cholesterol and DPhPC/cholesterol samples are presented. If cholesterol is present in moderate concentrations, it can substantially promote the formation of stalks at higher degree of hydration. In addition, a possibly new phase in DOPC/cholesterol is found at high cholesterol content in the low humidity range.

  8. Association of the pr Peptides with Dengue Virus at Acidic pH Blocks Membrane Fusion

    SciTech Connect

    Yu, I.-M.; Holdaway, H.A.; Chipman, P.R.; Kuhn, R.J.; Rossmann, M.G.; Chen, J.; Purdue

    2010-07-27

    Flavivirus assembles into an inert particle that requires proteolytic activation by furin to enable transmission to other hosts. We previously showed that immature virus undergoes a conformational change at low pH that renders it accessible to furin (I. M. Yu, W. Zhang, H. A. Holdaway, L. Li, V. A. Kostyuchenko, P. R. Chipman, R. J. Kuhn, M. G. Rossmann, and J. Chen, Science 319:1834-1837, 2008). Here we show, using cryoelectron microscopy, that the structure of immature dengue virus at pH 6.0 is essentially the same before and after the cleavage of prM. The structure shows that after cleavage, the proteolytic product pr remains associated with the virion at acidic pH, and that furin cleavage by itself does not induce any major conformational changes. We also show by liposome cofloatation experiments that pr retention prevents membrane insertion, suggesting that pr is present on the virion in the trans-Golgi network to protect the progeny virus from fusion within the host cell.

  9. Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion.

    PubMed

    Halldorsson, Steinar; Behrens, Anna-Janina; Harlos, Karl; Huiskonen, Juha T; Elliott, Richard M; Crispin, Max; Brennan, Benjamin; Bowden, Thomas A

    2016-06-28

    An emergent viral pathogen termed severe fever with thrombocytopenia syndrome virus (SFTSV) is responsible for thousands of clinical cases and associated fatalities in China, Japan, and South Korea. Akin to other phleboviruses, SFTSV relies on a viral glycoprotein, Gc, to catalyze the merger of endosomal host and viral membranes during cell entry. Here, we describe the postfusion structure of SFTSV Gc, revealing that the molecular transformations the phleboviral Gc undergoes upon host cell entry are conserved with otherwise unrelated alpha- and flaviviruses. By comparison of SFTSV Gc with that of the prefusion structure of the related Rift Valley fever virus, we show that these changes involve refolding of the protein into a trimeric state. Reverse genetics and rescue of site-directed histidine mutants enabled localization of histidines likely to be important for triggering this pH-dependent process. These data provide structural and functional evidence that the mechanism of phlebovirus-host cell fusion is conserved among genetically and patho-physiologically distinct viral pathogens.

  10. Structure of a phleboviral envelope glycoprotein reveals a consolidated model of membrane fusion.

    PubMed

    Halldorsson, Steinar; Behrens, Anna-Janina; Harlos, Karl; Huiskonen, Juha T; Elliott, Richard M; Crispin, Max; Brennan, Benjamin; Bowden, Thomas A

    2016-06-28

    An emergent viral pathogen termed severe fever with thrombocytopenia syndrome virus (SFTSV) is responsible for thousands of clinical cases and associated fatalities in China, Japan, and South Korea. Akin to other phleboviruses, SFTSV relies on a viral glycoprotein, Gc, to catalyze the merger of endosomal host and viral membranes during cell entry. Here, we describe the postfusion structure of SFTSV Gc, revealing that the molecular transformations the phleboviral Gc undergoes upon host cell entry are conserved with otherwise unrelated alpha- and flaviviruses. By comparison of SFTSV Gc with that of the prefusion structure of the related Rift Valley fever virus, we show that these changes involve refolding of the protein into a trimeric state. Reverse genetics and rescue of site-directed histidine mutants enabled localization of histidines likely to be important for triggering this pH-dependent process. These data provide structural and functional evidence that the mechanism of phlebovirus-host cell fusion is conserved among genetically and patho-physiologically distinct viral pathogens. PMID:27325770

  11. Membrane perturbation: studies employing a calcium-sensitive dye, arsenazo III, in liposomes.

    PubMed Central

    Weissmann, G; Collins, T; Evers, A; Dunham, P

    1976-01-01

    A metallochromic dye, arsenazo III [2,7-bis-(2-arsonophenylazo)-1,8-dihydroxynaphthalene-3,6-disulfonic acid], has been incorporated into the aquenous interspaces of multilamellar liposomes. multilamellar liposomes. Addition of Ca produced no shift in the absorbance spectrum of dye captured by liposomes, whereas disruption of liposomes by Triton X-100, followed by Ca, produced the spectrum chracteristic of the dye-Ca complex: evidence of latency. Addition of excess ethyleneglycol-bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) reversed the spectal shift. Differences between spectra obtained in this sequence yielded dye efflus. To measure Ca efflux, difference spectra (+/-EGTA) were obtained from cationic liposomes containing Ca after detergent lysis (sensitivity less than 10 mmol/ml). Since liposomes were impermeable either to dye or Ca until perturbed, it was possible to test a variety of membrane-active steroids (diethylstilbesterol, deoxycorticosterone, etiocholanolone) for their capacity to provoke dye efflux from liposomes; preincorporation of cortisol stablized liposomes against dye leak. Immunoglobulin-coated liposomes containing dye were taken up by phagocytes of Mustelus canis, and phagocytic vacuoles stained red-purple after ingestions. Liposomes containing the calcium-sensitive dye constitute a simple, accurate means for determining membrane perturbation and Ca fluxes; their uptake by cells or organelles remains to be exploited further. PMID:1061152

  12. A novel tridentate bis(phosphinic acid)phosphine oxide based europium(III)-selective Nafion membrane luminescent sensor.

    PubMed

    Sainz-Gonzalo, F J; Popovici, C; Casimiro, M; Raya-Barón, A; López-Ortiz, F; Fernández, I; Fernández-Sánchez, J F; Fernández-Gutiérrez, A

    2013-10-21

    A new europium(III) membrane luminescent sensor based on a new tridentate bis(phosphinic acid)phosphine oxide (3) system has been developed. The synthesis of this new ligand is described and its full characterization by NMR, IR and elemental analyses is provided. The luminescent complex formed between europium(III) chloride and ligand 3 was evaluated in solution, observing that its spectroscopic and chemical characteristics are excellent for measuring in polymer inclusion membranes. Included in a Nafion membrane, all the parameters (ligand and ionic additives) that can affect the sensitivity and selectivity of the sensing membrane as well as the instrumental conditions were carefully optimized. The best luminescence signal (λexc = 229.06 nm and λem = 616.02 nm) was exhibited by the sensing film having a Nafion : ligand composition of 262.3 : 0.6 mg mL(-1). The membrane sensor showed a short response time (t95 = 5.0 ± 0.2 min) and an optimum working pH of 5.0 (25 mM acetate buffer solution). The membrane sensor manifested a good selectivity toward europium(III) ions with respect to other trivalent metals (iron, chromium and aluminium) and lanthanide(III) ions (lanthanum, samarium, terbium and ytterbium), although a small positive interference of terbium(III) ions was observed. It provided a linear range from 1.9 × 10(-8) to 5.0 × 10(-6) M with a very low detection limit (5.8 × 10(-9) M) and sensitivity (8.57 × 10(-7) a.u. per M). The applicability of this sensing film has been demonstrated by analyzing different kinds of spiked water samples obtaining recovery percentages of 95-97%.

  13. Polymeric Membrane Electrodes with Improved Fluoride Selectivity and Lifetime Based on Zr(IV)- and Al(III)- Tetraphenylporphyrin Derivatives

    PubMed Central

    Pietrzak, Mariusz; Meyerhoff, Mark E.; Malinowska, Elżbieta

    2007-01-01

    Novel aluminum(III)- and zirconium(IV)-tetraphenylporhyrin (TPP) derivatives are examined as fluoride selective ionophores for preparing polymer membrane-based ion-selective electrodes (ISEs). The influence of t-butyl— or dichloro— phenyl ring substituents as well as the nature of the metal ion center (Al(III) vs. Zr(IV)) on the anion complexation constants of TPP derivative ionophores are reported. The anion binding stability constants of the ionophores are characterized by the so-called “sandwich membrane” method. All of the metalloporphyrins examined form their strongest anion complexes with fluoride. The influence of plasticizer as well as the type of lipophilic ionic site additive and their amounts in the sensing membrane are discussed. It is shown that membrane electrodes formulated with the metalloporphyrin derivatives and appropriate anionic or cationic additives exhibit enhanced potentiometric response toward fluoride over all other anions tested. Since selectivity toward fluoride is enhanced in the presence of both anionic and cationic additives, the metalloporphyrins can function as either charged or neutral carriers within the organic membrane phase. In contrast to previously reported fluoride-selective polymeric membrane electrodes based on metalloporphyrins, nernstian or near-nernstian (−51.2 to −60.1 mV decade−1) as well as rapid (t < 80s) and fully reversible potentiometric fluoride responses are observed. Moreover, use of aluminum(III)—t-butyltetraphenylporphyrin as the ionophore provides fluoride sensors with prolonged (7 months) functional life-time. PMID:17631098

  14. Accumulation of properly folded human type III procollagen molecules in specific intracellular membranous compartments in the yeast Pichia pastoris.

    PubMed

    Keizer-Gunnink, I; Vuorela, A; Myllyharju, J; Pihlajaniemi, T; Kivirikko, K I; Veenhuis, M

    2000-02-01

    It was recently reported that co-expression of the proalpha1(III) chain of human type III procollagen with the subunits of human prolyl 4-hydroxylase in Pichia pastoris produces fully hydroxylated and properly folded recombinant type III procollagen molecules (Vuorela, A., Myllyharju, J., Nissi, R., Pihlajaniemi, T., Kivirikko, K.I., 1997. Assembly of human prolyl 4-hydroxylase and type III collagen in the yeast Pichia pastoris: formation of a stable enzyme tetramer requires coexpression with collagen and assembly of a stable collagen requires coexpression with prolyl 4-hydroxylase. EMBO J. 16, 6702-6712). These properly folded molecules accumulated inside the yeast cell, however, only approximately 10% were found in the culture medium. We report here that replacement of the authentic signal sequence of the human proalpha1(III) with the Saccharomyces cerevisiae alpha mating factor prepro sequence led only to a minor increase in the amount secreted. Immunoelectron microscopy studies indicated that the procollagen molecules accumulate in specific membranous vesicular compartments that are closely associated with the nuclear membrane. Prolyl 4-hydroxylase, an endoplasmic reticulum (ER) lumenal enzyme, was found to be located in the same compartments. Non-helical proalpha1(III) chains produced by expression without recombinant prolyl 4-hydroxylase likewise accumulated within these compartments. The data indicate that properly folded recombinant procollagen molecules accumulate within the ER and do not proceed further in the secretory pathway. This may be related to the large size of the procollagen molecule. PMID:10686423

  15. Synthesis of CDP-diacylglycerol by rat liver rough microsomes as visualized by electron microscopic autoradiography: Relationship to GTP-stimulated membrane fusion

    SciTech Connect

    Jolicoeur, M.; Kan, F.W.; Paiement, J. )

    1991-03-01

    Following conditions of incubation for the analysis of liponucleotide synthesis, we compared GTP-dependent formation of CDP-diacylglycerol (CDP-DG) and membrane fusion in RNA-depleted rough microsomes from rat liver. After incubation of stripped rough microsomes (SRM) in the presence of GTP and (5-3H)-CTP, radioactivity was recovered in lipid extracts and identified by thin-layer chromatography as a single spot which co-migrated with CDP-DG. The nucleotide requirement for CDP-DG synthesis and that for membrane fusion were observed to be identical. We next carried out an electron microscopic autoradiographic analysis on incubated membranes to determine the site of incorporation of (5-3H)-CTP. Silver grains were observed directly over the unilamellar membranes of natural vesicles. In confirmation of the biochemical data, quantitation of silver grain density indicated more grains over membranes incubated in the presence of GTP than over those incubated in the absence of this nucleotide. For membranes incubated in the presence of GTP, the grain density was similar over fused and unfused membranes in the same preparation. When SRM were incubated with the enzyme co-factors required for synthesis of phosphatidylinositol, a GTP-independent membrane fusion was observed by both transmission and freeze-fracture electron microscopy. Together with the biochemical and autoradiographic data, this suggests that phospholipid metabolism may be activated by GTP and lead to the fusion of RER membrane.

  16. Membrane-Dependent Conformation, Dynamics, and Lipid Interactions of the Fusion Peptide of the Paramyxovirus PIV5 from Solid-State NMR

    PubMed Central

    Yao, Hongwei; Hong, Mei

    2014-01-01

    The entry of enveloped viruses into cells requires protein-catalyzed fusion of the viral and cell membranes. The structure–function relation of a hydrophobic fusion peptide (FP) in viral fusion proteins is still poorly understood. We report magic-angle-spinning solid-state NMR results of the membrane-bound conformation, dynamics, and lipid interactions of the FP of the F protein of the paramyxovirus, parainfluenza virus 5 (PIV5). 13C chemical shifts indicate that the PIV5 FP structure depends on the composition of the phospholipid membrane: the peptide is α-helical in palmitoyloleoylphosphatidylglycerol-containing anionic membranes but mostly β-sheet in neutral phosphocholine membranes. Other environmental factors, including peptide concentration, cholesterol, membrane reconstitution protocol, and a Lys solubility tag, do not affect the secondary structure. The α-helical and β-sheet states exhibit distinct dynamics and lipid interactions. The β- sheet FP is immobilized, resides on the membrane surface, and causes significant membrane curvature. In contrast, the α-helical FP undergoes intermediate-timescale motion and maintains the lamellar order of the membrane. Two-dimensional 31P–1H correlation spectra show clear 31P–water cross peaks for anionic membranes containing the α-helical FP but weak or no 31P–water cross peak for neutral membranes containing the β-sheet FP. These results suggest that the β-sheet FP may be associated with high-curvature dehydrated fusion intermediates, while the α-helical state may be associated with the extended prehairpin state and the post-fusion state. Conformational plasticity is also a pronounced feature of the influenza and human immunodeficiency virus FPs, suggesting that these Gly-rich sequences encode structural plasticity to generate and sense different membrane morphologies. PMID:23183373

  17. Performance of a Single Assay for Both Type III and Type VI TMPRSS2:ERG Fusions in Noninvasive Prediction of Prostate Biopsy Outcome

    PubMed Central

    Clark, Jarrod P.; Munson, Kristofer W.; Gu, Jessie W.; Lamparska-Kupsik, Katarzyna; Chan, Kevin G.; Yoshida, Jeffrey S.; Kawachi, Mark H.; Crocitto, Laura E.; Wilson, Timothy G.; Feng, Ziding; Smith, Steven S.

    2010-01-01

    BACKGROUND TMPRSS2:ERG fusions are promising prostate cancer biomarkers. Because they can occur in multiple forms in a single cancer specimen, we developed a quantitative PCR test that detects both type III and type VI TMPRSS2:ERG fusions. The assay is quantified from a standard curve determined with a plasmid-cloned type III TMPRSS2:ERG fusion target. METHODS We collected expressed prostatic secretion (EPS) under an institutional review board–approved, blinded, prospective study from 74 patients undergoing transrectal ultrasound-guided biopsy for prostate cancer. We compared the characteristic performance of the test for type III and type VI TMPRSS2:ERG fusions in predicting biopsy outcome and distinguishing between high and low Gleason scores with similar tests for the expression of PCA3 and DNA methylation levels of the APC, RARB, RASSF1, and GSTP1 genes. We used logistic regression to analyze the effects of multiple biomarkers in linear combinations. RESULTS Each test provided a significant improvement in characteristic performance over baseline digital rectal examination (DRE) plus serum prostate-specific antigen (PSA); however, the test for type III and type VI TMPRSS2:ERG fusions yielded the best performance in predicting biopsy outcome [area under the curve (AUC) 0.823, 95% CI 0.728–0.919, P < 0.001] and Gleason grade >7 (AUC 0.844, 95% CI 0.740–0.948, P < 0.001). CONCLUSIONS Although each test appears to have diagnostic value, PSA plus DRE plus type III and type VI TMPRSS2:ERG provided the best diagnostic performance in EPS specimens. PMID:18948370

  18. Determination of the topology of endoplasmic reticulum membrane proteins using redox-sensitive green-fluorescence protein fusions.

    PubMed

    Tsachaki, Maria; Birk, Julia; Egert, Aurélie; Odermatt, Alex

    2015-07-01

    Membrane proteins of the endoplasmic reticulum (ER) are involved in a wide array of essential cellular functions. Identification of the topology of membrane proteins can provide significant insight into their mechanisms of action and biological roles. This is particularly important for membrane enzymes, since their topology determines the subcellular site where a biochemical reaction takes place and the dependence on luminal or cytosolic co-factor pools and substrates. The methods currently available for the determination of topology of proteins are rather laborious and require post-lysis or post-fixation manipulation of cells. In this work, we have developed a simple method for defining intracellular localization and topology of ER membrane proteins in living cells, based on the fusion of the respective protein with redox-sensitive green-fluorescent protein (roGFP). We validated the method and demonstrated that roGFP fusion proteins constitute a reliable tool for the study of ER membrane protein topology, using as control microsomal 11β-hydroxysteroid dehydrogenase (11β-HSD) proteins whose topology has been resolved, and comparing with an independent approach. We then implemented this method to determine the membrane topology of six microsomal members of the 17β-hydroxysteroid dehydrogenase (17β-HSD) family. The results revealed a luminal orientation of the catalytic site for three enzymes, i.e. 17β-HSD6, 7 and 12. Knowledge of the intracellular location of the catalytic site of these enzymes will enable future studies on their biological functions and on the role of the luminal co-factor pool.

  19. Molecular dynamics analysis of conformational change of paramyxovirus F protein during the initial steps of membrane fusion

    SciTech Connect

    Martin-Garcia, Fernando; Mendieta-Moreno, Jesus Ignacio; Mendieta, Jesus

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Initial conformational change of paramyxovirus F protein is caused only by mechanical forces. Black-Right-Pointing-Pointer HRA region undergoes a structural change from a beta + alpha conformation to an extended coil and then to an all-alpha conformation. Black-Right-Pointing-Pointer HRS domains of F protein form three single {alpha}-helices prior to generation of the coiled coil. -- Abstract: The fusion of paramyxovirus to the cell membrane is mediated by fusion protein (F protein) present in the virus envelope, which undergoes a dramatic conformational change during the process. Unlike hemagglutinin in orthomyxovirus, this change is not mediated by an alteration of environmental pH, and its cause remains unknown. Steered molecular dynamics analysis leads us to suggest that the conformational modification is mediated only by stretching mechanical forces once the transmembrane fusion peptide of the protein is anchored to the cell membrane. Such elongating forces will generate major secondary structure rearrangement in the heptad repeat A region of the F protein; from {beta}-sheet conformation to an elongated coil and then spontaneously to an {alpha}-helix. In addition, it is proposed that the heptad repeat A region adopts a final three-helix coiled coil and that this structure appears after the formation of individual helices in each monomer.

  20. Development of new cloning vectors for the production of immunogenic outer membrane fusion proteins in Escherichia coli

    SciTech Connect

    Cornelis, P.; Sierra, J.C.; Lim, A. Jr.; Malur, A.

    1996-02-01

    The Pseudomonas aeruginosa lipoprotein gene (oprI) was modified by cloning an in-frame polylinker in both orientations at the end of oprI. The resulting plasmids pVUBI and pVUB2 allow high lipoprotein production in E. coli after IPTG induction. The modified lipoproteins are present in the outer membrane and surface-exposed. Outer membrane-bound fusion proteins of different sizes were produced and used to generate antibodies without use of adjuvant. An 87 bp DNA fragment from the vp72 capsid protein gene of African Swine Fever virus (ASFV) and the entire Leishmania major glycoprotein gp63 gene were expressed in this system. Finally, a fusion lipoprotein containing a 16 amino acid epitope from the preS2b region of Hepatitis B virus (HBV) was presented by an antigen-presenting cell line to a T-cell hybridoma while the corresponding cross-linked S2b peptide was not. The results suggest that OprI-based fusion proteins can be used to generate both humoral and cellular immune responses. 44 refs., 7 figs.

  1. Detection of closed influenza virus hemagglutinin fusion peptide structures in membranes by backbone (13)CO- (15)N rotational-echo double-resonance solid-state NMR.

    PubMed

    Ghosh, Ujjayini; Xie, Li; Weliky, David P

    2013-02-01

    The influenza virus fusion peptide is the N-terminal ~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 (13)CO-(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 (13)C shifts.

  2. Ca2+ and membrane binding to annexin 3 modulate the structure and dynamics of its N terminus and domain III

    PubMed Central

    Sopkova, Jana; Raguenes-Nicol, Céline; Vincent, Michel; Chevalier, Anne; Lewit-Bentley, Anita; Russo-Marie, Françoise; Gallay, Jacques

    2002-01-01

    Annexin 3 (ANX A3) represents ∼1% of the total protein of human neutrophils and promotes tight contact between membranes of isolated specific granules in vitro leading to their aggregation. Like for other annexins, the primary molecular events of the action of this protein is likely its binding to negatively charged phospholipid membranes in a Ca2+-dependent manner, via Ca2+-binding sites located on the convex side of the highly conserved core of the molecule. The conformation and dynamics of domain III can be affected by this process, as it was shown for other members of the family. The 20 amino-acid, N-terminal segment of the protein also could be affected and also might play a role in the modulation of its binding to the membranes. The structure and dynamics of these two regions were investigated by fluorescence of the two tryptophan residues of the protein (respectively, W190 in domain III and W5 in the N-terminal segment) in the wild type and in single-tryptophan mutants. By contrast to ANX A5, which shows a closed conformation and a buried W187 residue in the absence of Ca2+, domain III of ANX A3 exhibits an open conformation and a widely solvent-accessible W190 residue in the same conditions. This is in agreement with the three-dimensional structure of the ANX A3-E231A mutant lacking the bidentate Ca2+ ligand in domain III. Ca2+ in the millimolar concentration range provokes nevertheless a large mobility increase of the W190 residue, while interaction with the membranes reduces it slightly. In the N-terminal region, the W5 residue, inserted in the central pore of the protein, is weakly accessible to the solvent and less mobile than W190. Its amplitude of rotation increases upon binding of Ca2+ and returns to its original value when interacting with membranes. Ca2+ concentration for half binding of the W5A mutant to negatively charged membranes is ∼0.5 mM while it increases to ∼1 mM for the ANX A3 wild type and to ∼3 mM for the W190 ANX A3 mutant. In

  3. Outer membrane-associated serine protease involved in adhesion of Shewanella oneidensis to Fe(III) oxides.

    PubMed

    Burns, Justin L; Ginn, Brian R; Bates, David J; Dublin, Steven N; Taylor, Jeanette V; Apkarian, Robert P; Amaro-Garcia, Samary; Neal, Andrew L; Dichristina, Thomas J

    2010-01-01

    The facultative anaerobe Shewanella oneidensis MR-1 respires a variety of anaerobic electron acceptors, including insoluble Fe(III) oxides. S. oneidensis employs a number of novel strategies for respiration of insoluble Fe(III) oxides, including localization of respiratory proteins to the cell outer membrane (OM). The molecular mechanism by which S. oneidensis adheres to and respires Fe(III) oxides, however, remains poorly understood. In the present study, whole cell fractionation and MALDI-TOF-MS/MS techniques were combined to identify a serine protease (SO3800) associated with the S. oneidensis OM. SO3800 contained predicted structural motifs similar to cell surface-associated serine proteases that function as bacterial adhesins in other gram-negative bacteria. The gene encoding SO3800 was deleted from the S. oneidensis genome, and the resulting mutant strain (DeltaSO3800) was tested for its ability to adhere to and respire Fe(III) oxides. DeltaSO3800 was severely impaired in its ability to adhere to Fe(III) oxides, yet retained wild-type Fe(III) respiratory capability. Laser Doppler velocimetry and cryoetch high-resolution SEM experiments indicated that DeltaSO3800 displayed a lower cell surface charge and higher amount of surface-associated exopolysaccharides. Results of this study indicate that S. oneidensis may respire insoluble Fe(III) oxides at a distance, negating the requirement for attachment prior to electron transfer.

  4. Conservation of proteo-lipid nuclear membrane fusion machinery during early embryogenesis.

    PubMed

    Byrne, Richard D; Veeriah, Selvaraju; Applebee, Christopher J; Larijani, Banafshé

    2014-01-01

    The fusogenic lipid diacylglycerol is essential for remodeling gamete and zygote nuclear envelopes (NE) during early embryogenesis. It is unclear whether upstream signaling molecules are likewise conserved. Here we demonstrate PLCγ and its activator SFK1, which co-operate during male pronuclear envelope formation, also promote the subsequent male and female pronuclear fusion. PLCγ and SFK1 interact directly at the fusion site leading to PLCγ activation. This is accompanied by a spatially restricted reduction of PtdIns(4,5)P2. Consequently, pronuclear fusion is blocked by PLCγ or SFK1 inhibition. These findings identify new regulators of events in the early embryo and suggest a conserved "toolkit" of fusion machinery drives successive NE fusion events during embryogenesis.

  5. A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

    NASA Astrophysics Data System (ADS)

    Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

    2016-10-01

    An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ~10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

  6. A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

    PubMed Central

    Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

    2016-01-01

    An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ∼10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures. PMID:27708275

  7. Surface density of the Hendra G protein modulates Hendra F protein-promoted membrane fusion: role for Hendra G protein trafficking and degradation.

    PubMed

    Whitman, Shannon D; Dutch, Rebecca Ellis

    2007-07-01

    Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F.

  8. Surface density of the Hendra G protein modulates Hendra F protein-promoted membrane fusion: Role for Hendra G protein trafficking and degradation

    SciTech Connect

    Whitman, Shannon D.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2007-07-05

    Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F.

  9. Computer detection of the rapid diffusion of fluorescent membrane fusion markers in images observed with video microscopy.

    PubMed Central

    Niles, W D; Li, Q; Cohen, F S

    1992-01-01

    We have developed an algorithm for automated detection of the dynamic pattern characterizing flashes of fluorescence in video images of membrane fusion. The algorithm detects the spatially localized, transient increases and decreases in brightness that result from the dequenching of fluorescent dye in phospholipid vesicles or lipid-enveloped virions fusing with a planar membrane. The flash is identified in video images by its nonzero time derivative and the symmetry of its spatial profile. Differentiation is implemented by forward and backward subtractions of video frames. The algorithm groups spatially connected pixels brighter than a user-specified threshold into distinct objects in forward- and backward-differentiated images. Objects are classified as either flashes or noise particles by comparing the symmetries of matched forward and backward difference profiles and then by tracking each profile in successive difference images. The number of flashes identified depends on the brightness threshold, the size of the convolution kernel used to filter the image, and the time difference between the subtracted video frames. When these parameters are changed so that the algorithm identifies an increasing percentage of the flashes recognized by eye, an increasing number of noise objects are mistakenly identified as flashes. These mistaken flashes can be eliminated by a human observer. The algorithm considerably shortens the time needed to analyze video data. Tested extensively with phospholipid vesicle and virion fusion with planar membranes, our implementation of the algorithm accurately determined the rate of fusion of influenza virions labeled with the lipophilic dye octadecylrhodamine (R18). Images FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 6 PMID:1420909

  10. Effects of Mutations in the Rubella Virus E1 Glycoprotein on E1-E2 Interaction and Membrane Fusion Activity

    PubMed Central

    Yang, Decheng; Hwang, Dorothy; Qiu, Zhiyong; Gillam, Shirley

    1998-01-01

    Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer. PMID:9765418

  11. The conserved glycine-rich segment linking the N-terminal fusion peptide to the coiled coil of human T-cell leukemia virus type 1 transmembrane glycoprotein gp21 is a determinant of membrane fusion function.

    PubMed

    Wilson, Kirilee A; Bär, Séverine; Maerz, Anne L; Alizon, Marc; Poumbourios, Pantelis

    2005-04-01

    Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an approximately 90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment. PMID:15767455

  12. ESCRT-III binding protein MITD1 is involved in cytokinesis and has an unanticipated PLD fold that binds membranes

    PubMed Central

    Hadders, Michael A.; Agromayor, Monica; Obita, Takayuki; Perisic, Olga; Caballe, Anna; Kloc, Magdalena; Lamers, Meindert H.; Williams, Roger L.; Martin-Serrano, Juan

    2012-01-01

    The endosomal sorting complexes required for transport (ESCRT) proteins have a critical function in abscission, the final separation of the daughter cells during cytokinesis. Here, we describe the structure and function of a previously uncharacterized ESCRT-III interacting protein, MIT-domain containing protein 1 (MITD1). Crystal structures of MITD1 reveal a dimer, with a microtubule-interacting and trafficking (MIT) domain at the N terminus and a unique, unanticipated phospholipase D-like (PLD) domain at the C terminus that binds membranes. We show that the MIT domain binds to a subset of ESCRT-III subunits and that this interaction mediates MITD1 recruitment to the midbody during cytokinesis. Depletion of MITD1 causes a distinct cytokinetic phenotype consistent with destabilization of the midbody and abscission failure. These results suggest a model whereby MITD1 coordinates the activity of ESCRT-III during abscission with earlier events in the final stages of cell division. PMID:23045692

  13. The yeast vacuolar ABC transporter Ybt1p regulates membrane fusion through Ca2+ transport modulation

    PubMed Central

    Sasser, Terry L.; Padolina, Mark; Fratti, Rutilio A.

    2013-01-01

    Ybt1p is a class C ABC transporter (ATP-binding cassette transporter) that is localized to the vacuole of Saccharomyces cerevisiae. Although Ybt1p was originally identified as a bile acid transporter, it has also been found to function in other capacities, including the translocation of phosphatidylcholine to the vacuole lumen, and the regulation of Ca2+ homoeostasis. In the present study we found that deletion of YBT1 enhanced in vitro homotypic vacuole fusion by up to 50 % relative to wild-type vacuoles. The increased vacuole fusion was not due to aberrant protein sorting of SNAREs (soluble N-ethylmaleimide-sensitive factor-attachment protein receptors) or recruitment of factors from the cytosol such as Ypt7p and the HOPS (homotypic fusion and vacuole protein sorting) tethering complex. In addition, ybt1Δ vacuoles displayed no observable differences in the formation of SNARE complexes, interactions between SNAREs and HOPS, or formation of vertex microdomains. However, the absence of Ybt1p caused significant changes in Ca2+ transport during fusion. One difference was the prolonged Ca2+ influx exhibited by ybt1Δ vacuoles at the start of the fusion reaction. We also observed a striking delay in SNARE-dependent Ca2+ efflux. As vacuole fusion can be inhibited by high Ca2+ concentrations, we suggest that the delayed efflux in ybt1Δ vacuoles leads to the enhanced SNARE function. PMID:22970809

  14. Sensor fusion III: 3-D perception and recognition; Proceedings of the Meeting, Boston, MA, Nov. 5-8, 1990

    NASA Technical Reports Server (NTRS)

    Schenker, Paul S. (Editor)

    1991-01-01

    The volume on data fusion from multiple sources discusses fusing multiple views, temporal analysis and 3D motion interpretation, sensor fusion and eye-to-hand coordination, and integration in human shape perception. Attention is given to surface reconstruction, statistical methods in sensor fusion, fusing sensor data with environmental knowledge, computational models for sensor fusion, and evaluation and selection of sensor fusion techniques. Topics addressed include the structure of a scene from two and three projections, optical flow techniques for moving target detection, tactical sensor-based exploration in a robotic environment, and the fusion of human and machine skills for remote robotic operations. Also discussed are K-nearest-neighbor concepts for sensor fusion, surface reconstruction with discontinuities, a sensor-knowledge-command fusion paradigm for man-machine systems, coordinating sensing and local navigation, and terrain map matching using multisensing techniques for applications to autonomous vehicle navigation.

  15. Recruitment and SNARE-mediated fusion of vesicles in furrow membrane remodeling during cytokinesis in zebrafish embryos

    SciTech Connect

    Ming Liwai; Webb, Sarah E.; Lee, Karen W.; Miller, Andrew L. . E-mail: almiller@ust.hk

    2006-10-15

    Cytokinesis is the final stage in cell division that serves to partition cytoplasm and daughter nuclei into separate cells. Membrane remodeling at the cleavage plane is a required feature of cytokinesis in many species. In animal cells, however, the precise mechanisms and molecular interactions that mediate this process are not yet fully understood. Using real-time imaging in live, early stage zebrafish embryos, we demonstrate that vesicles labeled with the v-SNARE, VAMP-2, are recruited to the cleavage furrow during deepening in a microtubule-dependent manner. These vesicles then fuse with, and transfer their VAMP-2 fluorescent label to, the plasma membrane during both furrow deepening and subsequent apposition. This observation indicates that new membrane is being inserted during these stages of cytokinesis. Inhibition of SNAP-25 (a cognate t-SNARE of VAMP-2), using a monoclonal antibody, blocked VAMP-2 vesicle fusion and furrow apposition. Transient expression of mutant forms of SNAP-25 also produced defects in furrow apposition. SNAP-25 inhibition by either method, however, did not have any significant effect on furrow deepening. Thus, our data clearly indicate that VAMP-2 and SNAP-25 play an essential role in daughter blastomere apposition, possibly via the delivery of components that promote the cell-to-cell adhesion required for the successful completion of cytokinesis. Our results also support the idea that new membrane addition, which occurs during late stage cytokinesis, is not required for furrow deepening that results from contractile band constriction.

  16. Isotopically Labeled Expression in E. coli, Purification, and Refolding of the Full Ectodomain of the Influenza Virus Membrane Fusion Protein

    PubMed Central

    Curtis-Fisk, Jaime; Spencer, Ryan M.; Weliky, David P.

    2008-01-01

    This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of E. coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A600 ~ 8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the 13CO chemical shift measured using solid-state nuclear magnetic resonance spectroscopy. PMID:18640277

  17. Efficient isolation of Pseudomonas aeruginosa type III secretion translocators and assembly of heteromeric transmembrane pores in model membranes.

    PubMed

    Romano, Fabian B; Rossi, Kyle C; Savva, Christos G; Holzenburg, Andreas; Clerico, Eugenia M; Heuck, Alejandro P

    2011-08-23

    Translocation of bacterial toxins or effectors into host cells using the type III secretion (T3S) system is a conserved mechanism shared by many Gram-negative pathogens. Pseudomonas aeruginosa injects different proteins across the plasma membrane of target cells, altering the normal metabolism of the host. Protein translocation presumably occurs through a proteinaceous transmembrane pore formed by two T3S secreted protein translocators, PopB and PopD. Unfolded translocators are secreted through the T3S needle prior to insertion into the target membrane. Purified PopB and PopD form pores in model membranes. However, their tendency to form heterogeneous aggregates in solution had hampered the analysis of how these proteins undergo the transition from a denatured state to a membrane-inserted state. Translocators were purified as stable complexes with the cognate chaperone PcrH and isolated from the chaperone using 6 M urea. We report here the assembly of stable transmembrane pores by dilution of urea-denatured translocators in the presence of membranes. PopB and PopD spontaneously bound liposomes containing anionic phospholipids and cholesterol in a pH-dependent manner as observed by two independent assays, time-resolved Förster resonance energy transfer and sucrose-step gradient ultracentrifugation. Using Bodipy-labeled proteins, we found that PopB interacts with PopD on the membrane surface as determined by excitation energy migration and fluorescence quenching. Stable transmembrane pores are more efficiently assembled at pH <5.0, suggesting that acidic residues might be involved in the initial membrane binding and/or insertion. Altogether, the experimental setup described here represents an efficient method for the reconstitution and analysis of membrane-inserted translocators.

  18. Characterization of the pH-induced fusion of liposomes with the plasma membrane of rye protoplasts

    SciTech Connect

    Arvinte , T.; Steponkus, P.L.

    1988-07-26

    The authors present evidence that at acidic pH, liposomes composed of soybean lipids fuse with the plasma membrane of protoplasts isolated from rye leaves. Using the resonance energy transfer assay (RET), they determined the rate and extent of liposome and protoplast plasma membrane lipid mixing. The fluorescent donor-acceptor pair was N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidyl-ethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)phosphatidylethanolamine (N-Rh-PE). Fusion was substantial below pH 5, and the half-time of lipid mixing was fast (t/sub 1/2/ on the order of minutes) and pH, concentration, and temperature dependent. The extent of liposome and protoplast fusion from the total amount of liposomes associated with the protoplasts was also determined by the RET assay. Protoplasts were incubated with fluorescent-labeled liposomes (5 min at 30/sup 0/C) at different pH values and then washed twice by centrifugation. The fluorescence spectra of the protoplast suspension permitted determination of the ratio of N-NBD-PE emission at 530 nm to the N-Rh-PE emission at 590 nm, which is a measure of the degree of lipid mixing. The amount of liposomes associated (fused and unfused) with protoplasts at pH 3.9 was approximately 9 times greater than that at pH 5.6. The transfer of liposome contents to the protoplast interior was studied with a method based on the fluorescence enhancement of a solution of calcein, initially confined in the liposomes at self-quenching concentrations. The kinetics of calcein release were very similar to those of lipid mixing. Fluorescence microscopy showed that after fusion with liposomes containing calcein, the protoplasts exhibited a strong diffuse fluorescence in the interior.

  19. Construction of membrane-anchoring fusion protein of Thermococcus kodakaraensis glycerol kinase and its application to repetitive batchwise reactions.

    PubMed

    Restiawaty, Elvi; Honda, Kohsuke; Okano, Kenji; Hirota, Ryuichi; Omasa, Takeshi; Kuroda, Akio; Ohtake, Hisao

    2012-04-01

    We previously demonstrated the stoichiometric conversion of glycerol to glycerol-3-phosphate (G3P) using Escherichia coli recombinants producing the ATP-dependent glycerol kinase of the hyperthermophile Thermococcus kodakaraensis (TkGK) and the polyphosphate kinase of Thermus thermophilus HB27 (TtPPK). TtPPK was associated with the membrane fraction of E. coli recombinants, whereas TkGK was released from the cells during the reaction at 70°C. In this study, TkGK was fused with either TtPPK or an E. coli membrane-intrinsic protein, YedZ, to minimize the heat-induced leakage of TkGK. When the E. coli recombinants having these fusion proteins were incubated at 70°C for 2h, more than 80% of TkGK activity was retained in the heated E. coli cells. However, the yields of G3P production by E. coli having the fusion proteins of TtPPK and TkGK were only less than 35%. Polyphosphate is a strong chelator for metal ions and has an inhibitory effect on TkGK which requires magnesium. Insufficient space between TtPPK and TkGK might enhance the inhibitory effect of polyphosphate on TkGK activity of the fusion protein. The mixture of E. coli cells having TtPPK and those having TkGK fused with YedZ converted 80% of glycerol into G3P. These recombinant cells could be easily recovered from the reaction mixture by centrifugation and repeatedly used without a significant loss of enzyme activities.

  20. Is the optimal pH for membrane fusion in host cells by avian influenza viruses related to host range and pathogenicity?

    PubMed

    Okamatsu, Masatoshi; Motohashi, Yurie; Hiono, Takahiro; Tamura, Tomokazu; Nagaya, Kazuki; Matsuno, Keita; Sakoda, Yoshihiro; Kida, Hiroshi

    2016-08-01

    Influenza viruses isolated from wild ducks do not replicate in chickens. This fact is not explained solely by the receptor specificity of the hemagglutinin (HA) from such viruses for target host cells. To investigate this restriction in host range, the fusion activities of HA molecules from duck and chicken influenza viruses were examined. Influenza viruses A/duck/Mongolia/54/2001 (H5N2) (Dk/MNG) and A/chicken/Ibaraki/1/2005 (H5N2) (Ck/IBR), which replicate only in their primary hosts, were used. The optimal pH for membrane fusion of Ck/IBR was 5.9, higher than that of Dk/MNG at 4.9. To assess the relationship between the optimal pH for fusion and the host range of avian influenza viruses, the optimal pH for fusion of 55 influenza virus strains isolated from ducks and chickens was examined. No correlation was found between the host range and optimal pH for membrane fusion by the viruses, and this finding applied also to the H5N1 highly pathogenic avian influenza viruses. The optimal pH for membrane fusion for avian influenza viruses was shown to not necessarily be correlated with their host range or pathogenicity in ducks and chickens. PMID:27231009

  1. Heparin-glutathione III: study with fluorescent probes as indicators of membrane status of bull sperm.

    PubMed

    Reyes, R; Martinez, J C; Delgado, N M; Merchant-Larios, H

    2002-01-01

    Sperm obtained from bull epididymes were used to validate in vitro the effect of heparin and reduced glutathione on sperm membrane status, with the use of sodium dodecyl sulfate (SDS) and Triton X-100 in the presence of propidium iodide (IP) and diacetate fluorescein (FDA). The metabolic activities of treated sperm were qualitatively monitored using an alamar Blue Redox fluorescence indicator. Heparin did not damage the sperm plasma membrane, whereas GSH and SDS at 26 h of incubation dissolved the plasma membrane and the acrosome. On the other hand, at time zero, Triton X-100 showed 75% of sperm stained with IP, indicating plasma membrane damage. Results validated by electron microscopy of thin sections of treated sperm showed complete lack of the membrane, acrosome, and postacrosomal membrane system with 0.01% Triton X-100. Extracellular 15 mM GSH completely disappeared the plasma membrane over the sperm nucleus, leaving the postacrosomal membrane system and nucleus without apparent damage. The metabolic activity was supported over 52 h of incubation in any of the incubation systems tested, including Triton X-100, which showed a spermaticide effect. The authors propose that membrane damage does not mean they are dead, no matter the vital stain employed, and also that FDA-IP staining can be used as a fluorescent marker of sperm plasmatic membrane permeabilization and nuclear swelling.

  2. NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts.

    PubMed

    Baur, Tina; Ramadan, Kristijan; Schlundt, Andreas; Kartenbeck, Jürgen; Meyer, Hemmo H

    2007-08-15

    Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSF(E329Q) variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.

  3. Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters.

    PubMed

    Bird, Louise E; Nettleship, Joanne E; Järvinen, Valtteri; Rada, Heather; Verma, Anil; Owens, Raymond J

    2016-01-01

    The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years. PMID:27553231

  4. Closed and Semiclosed Interhelical Structures in Membrane vs Closed and Open Structures in Detergent for the Influenza Virus Hemagglutinin Fusion Peptide and Correlation of Hydrophobic Surface Area with Fusion Catalysis.

    PubMed

    Ghosh, Ujjayini; Xie, Li; Jia, Lihui; Liang, Shuang; Weliky, David P

    2015-06-24

    The ∼25 N-terminal "HAfp" residues of the HA2 subunit of the influenza virus hemagglutinin protein are critical for fusion between the viral and endosomal membranes at low pH. Earlier studies of HAfp in detergent support (1) N-helix/turn/C-helix structure at pH 5 with open interhelical geometry and N-helix/turn/C-coil structure at pH 7; or (2) N-helix/turn/C-helix at both pHs with closed interhelical geometry. These different structures led to very different models of HAfp membrane location and different models of catalysis of membrane fusion by HAfp. In this study, the interhelical geometry of membrane-associated HAfp is probed by solid-state NMR. The data are well-fitted to a population mixture of closed and semiclosed structures. The two structures have similar interhelical geometries and are planar with hydrophobic and hydrophilic faces. The different structures of HAfp in detergent vs membrane could be due to the differences in interaction with the curved micelle vs flat membrane with better geometric matching between the closed and semiclosed structures and the membrane. The higher fusogenicity of longer sequences and low pH is correlated with hydrophobic surface area and consequent increased membrane perturbation.

  5. pH-Dependent Formation and Disintegration of the Influenza A Virus Protein Scaffold To Provide Tension for Membrane Fusion

    PubMed Central

    Shilova, L. A.; Kachala, M. V.; Tashkin, V. Y.; Sokolov, V. S.; Fedorova, N. V.; Baratova, L. A.; Knyazev, D. G.; Zimmerberg, J.; Chizmadzhev, Y. A.

    2015-01-01

    ABSTRACT Influenza virus is taken up from a pH-neutral extracellular milieu into an endosome, whose contents then acidify, causing changes in the viral matrix protein (M1) that coats the inner monolayer of the viral lipid envelope. At a pH of ∼6, M1 interacts with the viral ribonucleoprotein (RNP) in a putative priming stage; at this stage, the interactions of the M1 scaffold coating the lipid envelope are intact. The M1 coat disintegrates as acidification continues to a pH of ∼5 to clear a physical path for the viral genome to transit from the viral interior to the cytoplasm. Here we investigated the physicochemical mechanism of M1's pH-dependent disintegration. In neutral media, the adsorption of M1 protein on the lipid bilayer was electrostatic in nature and reversible. The energy of the interaction of M1 molecules with each other in M1 dimers was about 10 times as weak as that of the interaction of M1 molecules with the lipid bilayer. Acidification drives conformational changes in M1 molecules due to changes in the M1 charge, leading to alterations in their electrostatic interactions. Dropping the pH from 7.1 to 6.0 did not disturb the M1 layer; dropping it lower partially desorbed M1 because of increased repulsion between M1 monomers still stuck to the membrane. Lipid vesicles coated with M1 demonstrated pH-dependent rupture of the vesicle membrane, presumably because of the tension generated by this repulsive force. Thus, the disruption of the vesicles coincident with M1 protein scaffold disintegration at pH 5 likely stretches the lipid membrane to the point of rupture, promoting fusion pore widening for RNP release. IMPORTANCE Influenza remains a top killer of human beings throughout the world, in part because of the influenza virus's rapid binding to cells and its uptake into compartments hidden from the immune system. To attack the influenza virus during this time of hiding, we need to understand the physical forces that allow the internalized virus to

  6. Dysferlin Binds SNAREs (Soluble N-Ethylmaleimide-sensitive Factor (NSF) Attachment Protein Receptors) and Stimulates Membrane Fusion in a Calcium-sensitive Manner.

    PubMed

    Codding, Sara J; Marty, Naomi; Abdullah, Nazish; Johnson, Colin P

    2016-07-01

    Resealing of tears in the sarcolemma of myofibers is a necessary step in the repair of muscle tissue. Recent work suggests a critical role for dysferlin in the membrane repair process and that mutations in dysferlin are responsible for limb girdle muscular dystrophy 2B and Miyoshi myopathy. Beyond membrane repair, dysferlin has been linked to SNARE-mediated exocytotic events including cytokine release and acid sphingomyelinase secretion. However, it is unclear whether dysferlin regulates SNARE-mediated membrane fusion. In this study we demonstrate a direct interaction between dysferlin and the SNARE proteins syntaxin 4 and SNAP-23. In addition, analysis of FRET and in vitro reconstituted lipid mixing assays indicate that dysferlin accelerates syntaxin 4/SNAP-23 heterodimer formation and SNARE-mediated lipid mixing in a calcium-sensitive manner. These results support a function for dysferlin as a calcium-sensing SNARE effector for membrane fusion events. PMID:27226605

  7. Influence of lipid composition on physical properties and peg-mediated fusion of curved and uncurved model membrane vesicles: "nature's own" fusogenic lipid bilayer.

    PubMed

    Haque, M E; McIntosh, T J; Lentz, B R

    2001-04-10

    Poly(ethylene glycol) (PEG)-mediated fusion of phosphatidylcholine model membranes has been shown to mimic the protein-mediated biomembrane process [Lee, J., and Lentz, B. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9274-9279]. Unlike the simple model membranes used in this earlier study, the lipid composition of fusogenic biomembranes is quite complex. The purpose of this paper was to examine PEG-mediated fusion of highly curved (SUV) and largely uncurved (LUV) membrane vesicles composed of different lipids in order to identify lipid compositions that produce highly fusogenic membranes. Starting with liposomes composed of five lipids with different physical properties, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), bovine brain sphingomyelin (SM), and cholesterol (CH), we systematically varied the composition and tested for the extent of PEG-mediated fusion after 5 min of treatment. We found that a vesicle system composed of four lipids, DOPC/DOPE/SM/CH, fused optimally at a 35/30/15/20 molar ratio. Each lipid seemed to play a part in optimizing the membrane for fusion. PE disrupted outer leaflet packing as demonstrated with TMA-DPH lifetime, C(6)-NBD-PC partitioning, and DPH anisotropy measurements, and thus significantly enhanced fusion and rupture, without significantly altering interbilayer approach (X-ray diffraction). An optimal ratio of PC/PE (35/30) produced a balance between fusion and rupture. CH and SM, when present at an optimal ratio of 3/4 in vesicles containing the optimal PC/PE ratio, reduced rupture without significantly reducing fusion. This optimal CH/SM ratio also enhanced outer leaflet packing, suggesting that fusion is dependent not only on outer leaflet packing but also on the properties of the inner leaflet. Addition of CH without SM enhanced rupture relative to fusion, while SM alone reduced both rupture and fusion. The optimal lipid composition is very close to the natural

  8. A Mechanistic Study of Arsenic (III) Rejection by Reverse Osmosis and Nanofiltration Membranes

    ERIC Educational Resources Information Center

    Suzuki, Tasuma

    2009-01-01

    Reverse osmosis/nanofiltration (RO/NF) membranes are capable to provide an effective barrier for a wide range of contaminants (including disinfection by-products precursors) in a single treatment step. However, solute rejection mechanisms by RO/NF membranes are not well understood. The lack of mechanistic information arises from experimental…

  9. Thermodynamics of Micelle Formation and Membrane Fusion Modulate Antimicrobial Lipopeptide Activity.

    PubMed

    Lin, Dejun; Grossfield, Alan

    2015-08-18

    Antimicrobial lipopeptides (AMLPs) are antimicrobial drug candidates that preferentially target microbial membranes. One class of AMLPs, composed of cationic tetrapeptides attached to an acyl chain, have minimal inhibitory concentrations in the micromolar range against a range of bacteria and fungi. Previously, we used coarse-grained molecular dynamics simulations and free energy methods to study the thermodynamics of their interaction with membranes in their monomeric state. Here, we extended the study to the biologically relevant micellar state, using, to our knowledge, a novel reaction coordinate based on hydrophobic contacts. Using umbrella sampling along this reaction coordinate, we identified the critical transition states when micelles insert into membranes. The results indicate that the binding of these AMLP micelles to membranes is thermodynamically favorable, but in contrast to the monomeric case, there are significant free energy barriers. The height of these free energy barriers depends on the membrane composition, suggesting that the AMLPs' ability to selectively target bacterial membranes may be as much kinetic as thermodynamic. This mechanism highlights the importance of considering oligomeric state in solution as criterion when optimizing peptides or lipopeptides as antibiotic leads.

  10. Display of E. coli Alkaline Phosphatase pIII or pVIII Fusions on Phagemid Surfaces Reveals Monovalent Decoration with Active Molecules

    PubMed Central

    Weichel, Michael; Jaussi, Rolf; Rhyner, Claudio; Crameri, Reto

    2008-01-01

    Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.1) was displayed via the leucine zipper element of the Jun-Fos heterodimer on the surface of filamentous phage and the kinetic parameters Km and kcat were determined. The phoA gene was cloned downstream of fos while jun was inserted upstream of pIII or pVIII, alternatively, in the pJuFo phagemid vector. Both fusion genes are regulated by independent lacZ promoters. PhoA displayed on the phagemid pIII surface exhibited a Km of 11.2 µM with 4-nitrophenyl phosphate as substrate, which is consistent with data published for soluble PhoA. Based on these data we calculated the decoration of pJuFo phagemid with PhoA using the minor and major coat proteins pIII and pVIII as fusion partners under variable inducing conditions. We found that, even if the promoters are fully induced at a concentration of 1000 µM IPTG, the phagemids display maximally one copy of PhoA-Fos-Jun-coat protein fusion, irrespective of whether the protein is presented via pIII or pVIII. However, since PhoA is displayed in a native-like fashion, as deduced from the kinetic parameters of the enzymatic reaction, the pJuFo technology provides a versatile tool for the functional screening of complex cDNA libraries displayed on the phagemids' surface. PMID:18949073

  11. Selectivity Enhancement for Chloride Ion by In(III)-Porphyrin-Based Polymeric Membrane Electrode Operated in Pulsed Chronopotentiometric Mode

    PubMed Central

    Gemene, Kebede L.; Meyerhoff, Mark E.

    2013-01-01

    A robust selectivity enhancement of an In(III)-porphyrin ionophore-based chloride-selective electrode under pulsed chronopotentiometric measurement mode that enables the detection of chloride ions in the presence of a normally interfering concentration of salicylate ions is described. This enhancement is achieved by the rapid depletion of the surface concentration of the more dilute lipophilic anion during an initial anodic current pulse period due to extraction of this preferred anion into the membrane phase. Measurement of chloride with a detection limit of 8 mM and near Nernstian response slope in the presence of 1 mM salicylate is possible using the pulstrode method. PMID:23355767

  12. Apolipophorin III interaction with model membranes composed of phosphatidylcholine and sphingomyelin using differential scanning calorimetry.

    PubMed

    Chiu, Michael H; Wan, Chung-Ping Leon; Weers, Paul M M; Prenner, Elmar J

    2009-10-01

    Apolipophorin III (apoLp-III) from Locusta migratoria was employed as a model apolipoprotein to gain insight into binding interactions with lipid vesicles. Differential scanning calorimetry (DSC) was used to measure the binding interaction of apoLp-III with liposomes composed of mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SM). Association of apoLp-III with multilamellar liposomes occurred over a temperature range around the liquid crystalline phase transition (L(alpha)). Qualitative and quantitative data were obtained from changes in the lipid phase transition upon addition of apoLp-III. Eleven ratios of DMPC and SM were tested from pure DMPC to pure SM. Broadness of the phase transition (T(1/2)), melting temperature of the phase transition (T(m)) and enthalpy were used to determine the relative binding affinity to the liposomes. Multilamellar vesicles composed of 40% DMPC and 60% SM showed the greatest interaction with apoLp-III, indicated by large T(1/2) values. Pure DMPC showed the weakest interaction and liposomes with lower percentage of DMPC retained domains of pure DMPC, even upon apoLp-III binding indicating demixing of liposome lipids. Addition of apoLp-III to rehydrated liposomes was compared to codissolved trials, in which lipids were rehydrated in the presence of protein, forcing the protein to interact with the lipid system. Similar trends between the codissolved and non-codissolved trials were observed, indicating a similar binding affinity except for pure DMPC. These results suggested that surface defects due to non-ideal packing that occur at the phase transition temperature of the lipid mixtures are responsible for apolipoprotein-lipid interaction in DMPC/SM liposomes. PMID:19647717

  13. Key steps in type III secretion system (T3SS) towards translocon assembly with potential sensor at plant plasma membrane.

    PubMed

    Ji, Hongtao; Dong, Hansong

    2015-09-01

    Many plant- and animal-pathogenic Gram-negative bacteria employ the type III secretion system (T3SS) to translocate effector proteins from bacterial cells into the cytosol of eukaryotic host cells. The effector translocation occurs through an integral component of T3SS, the channel-like translocon, assembled by hydrophilic and hydrophobic proteinaceous translocators in a two-step process. In the first, hydrophilic translocators localize to the tip of a proteinaceous needle in animal pathogens, or a proteinaceous pilus in plant pathogens, and associate with hydrophobic translocators, which insert into host plasma membranes in the second step. However, the pilus needs to penetrate plant cell walls in advance. All hydrophilic translocators so far identified in plant pathogens are characteristic of harpins: T3SS accessory proteins containing a unitary hydrophilic domain or an additional enzymatic domain. Two-domain harpins carrying a pectate lyase domain potentially target plant cell walls and facilitate the penetration of the pectin-rich middle lamella by the bacterial pilus. One-domain harpins target plant plasma membranes and may play a crucial role in translocon assembly, which may also involve contrapuntal associations of hydrophobic translocators. In all cases, sensory components in the target plasma membrane are indispensable for the membrane recognition of translocators and the functionality of the translocon. The conjectural sensors point to membrane lipids and proteins, and a phosphatidic acid and an aquaporin are able to interact with selected harpin-type translocators. Interactions between translocators and their sensors at the target plasma membrane are assumed to be critical for translocon assembly.

  14. Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity.

    PubMed

    Chen, Chen; Zhao, Guozhong; Chen, Wei; Guo, Benheng

    2015-11-01

    Although fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a β-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome- and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum.

  15. Metabolism of Fructooligosaccharides in Lactobacillus plantarum ST-III via Differential Gene Transcription and Alteration of Cell Membrane Fluidity.

    PubMed

    Chen, Chen; Zhao, Guozhong; Chen, Wei; Guo, Benheng

    2015-11-01

    Although fructooligosaccharides (FOS) can selectively stimulate the growth and activity of probiotics and beneficially modulate the balance of intestinal microbiota, knowledge of the molecular mechanism for FOS metabolism by probiotics is still limited. Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth of Lactobacillus plantarum ST-III using FOS or glucose as the sole carbon source. A total of 363 genes were differentially transcribed; in particular, two gene clusters were induced by FOS. Gene inactivation revealed that both of the clusters participated in the metabolism of FOS, which were transported across the membrane by two phosphotransferase systems (PTSs) and were subsequently hydrolyzed by a β-fructofuranosidase (SacA) in the cytoplasm. Combining the measurements of the transcriptome- and membrane-related features, we discovered that the genes involved in the biosynthesis of fatty acids (FAs) were repressed in cells grown on FOS; as a result, the FA profiles were altered by shortening of the carbon chains, after which membrane fluidity increased in response to FOS transport and utilization. Furthermore, incremental production of acetate was observed in both the transcriptomic and the metabolic experiments. Our results provided new insights into gene transcription, the production of metabolites, and membrane alterations that could explain FOS metabolism in L. plantarum. PMID:26319882

  16. Exploring the membrane fusion mechanism through force-induced disassembly of HIV-1 six-helix bundle.

    PubMed

    Gao, Kai; Zhang, Yong; Lou, Jizhong

    2016-05-13

    Enveloped virus, such as HIV-1, employs membrane fusion mechanism to invade into host cell. HIV-1 gp41 ectodomain uses six-helix bundle configuration to accomplish this process. Using molecular dynamic simulations, we confirmed the stability of this six-helix bundle by showing high occupancy of hydrogen bonds and hydrophobic interactions. Key residues and interactions important for the bundle integration were characterized by force-induced unfolding simulations of six-helix bundle, exhibiting the collapse order of these groups of interactions. Moreover, our results in some way concerted with a previous theory that the formation of coiled-coil choose a route which involved cooperative interactions between the N-terminal and C-terminal helix.

  17. F-actin-myosin II inhibitors affect chromaffin granule plasma membrane distance and fusion kinetics by retraction of the cytoskeletal cortex.

    PubMed

    Villanueva, José; Torres, Vanesa; Torregrosa-Hetland, Cristina J; Garcia-Martinez, Virginia; López-Font, Inmaculada; Viniegra, Salvador; Gutiérrez, Luis M

    2012-10-01

    Chromaffin cell catecholamines are released when specialized secretory vesicles undergo exocytotic membrane fusion. Evidence indicates that vesicle supply and fusion are controlled by the activity of the cortical F-actin-myosin II network. To study in detail cell cortex and vesicle interactions, we use fluorescent labeling with GFP-lifeact and acidotropic dyes in confocal and evanescent wave microscopy. These techniques provide structural details and dynamic images of chromaffin granules caged in a complex cortical structure. Both the movement of cortical structures and granule motion appear to be linked, and this motion can be restricted by the myosin II-specific inhibitor, blebbistatin, and the F-actin stabilizer, jasplakinolide. These treatments also affect the position of the vesicles in relation to the plasma membrane, increasing the distance between them and the fusion sites. Consequently, we observed slower single vesicle fusion kinetics in treated cells after neutralization of acridine orange-loaded granules during exocytosis. Increasing the distance between the granules and the fusion sites appears to be linked to the retraction of the F-actin cytoskeleton when treated with jasplakinolide. Thus, F-actin-myosin II inhibitors appear to slow granule fusion kinetics by altering the position of vesicles after relaxation of the cortical network.

  18. Force-induced globule-coil transition in laminin binding protein and its role for viral-cell membrane fusion.

    PubMed

    Zaitsev, Boris N; Benedetti, Fabrizio; Mikhaylov, Andrey G; Korneev, Denis V; Sekatskii, Sergey K; Karakouz, Tanya; Belavin, Pavel A; Netesova, Nina A; Protopopova, Elena V; Konovalova, Svetlana N; Dietler, Giovanni; Loktev, Valery B

    2014-12-01

    The specific interactions of the pairs laminin binding protein (LBP)-purified tick-borne encephalitis viral surface protein E and certain recombinant fragments of this protein, as well as West Nile viral surface protein E and certain recombinant fragments of that protein, are studied by combined methods of single-molecule dynamic force spectroscopy (SMDFS), enzyme immunoassay and optical surface waves-based biosensor measurements. The experiments were performed at neutral pH (7.4) and acid pH (5.3) conditions. The data obtained confirm the role of LBP as a cell receptor for two typical viral species of the Flavivirus genus. A comparison of these data with similar data obtained for another cell receptor of this family, namely human αVβ3 integrin, reveals that both these receptors are very important. Studying the specific interaction between the cell receptors in question and specially prepared monoclonal antibodies against them, we could show that both interaction sites involved in the process of virus-cell interaction remain intact at pH 5.3. At the same time, for these acid conditions characteristic for an endosome during flavivirus-cell membrane fusion, SMDFS data reveal the existence of a force-induced (effective already for forces as small as 30-70 pN) sharp globule-coil transition for LBP and LBP-fragments of protein E complexes. We argue that this conformational transformation, being an analog of abrupt first-order phase transition and having similarity with the famous Rayleigh hydrodynamic instability, might be indispensable for the flavivirus-cell membrane fusion process. PMID:25319621

  19. Application of REDOR subtraction for filtered MAS observation of labeled backbone carbons of membrane-bound fusion peptides

    NASA Astrophysics Data System (ADS)

    Yang, Jun; Parkanzky, Paul D.; Bodner, Michele L.; Duskin, Craig A.; Weliky, David P.

    2002-12-01

    Clean MAS observation of 13C-labeled carbons in membrane-bound HIV-1 and influenza fusion peptides was made by using a rotational-echo double-resonance spectroscopy (REDOR) filter of directly bonded 13C- 15N pairs. The clean filtering achieved with the REDOR approach is superior to filtering done with sample difference spectroscopy. In one labeling approach, the peptide had labels at a single 13C carbonyl and its directly bonded 15N. The resulting chemical shift distribution of the filtered signal is used to assess the distribution of local secondary structures at the labeled carbonyl. For the influenza peptide, the Leu-2 carbonyl chemical shift distribution is shown to vary markedly with lipid and detergent composition, as well as peptide:lipid ratio, suggesting that the local peptide structure also has a strong dependence on these factors. Because most carboxylic- and amino-labeled amino acids are commercially available, this REDOR approach should have broad applicability to chemically synthesized peptides as well as bacterially synthesized proteins. In a second labeling approach, the HIV-1 fusion peptide had U- 13C, 15N labeling over three sequential residues. When a 1.6 ms REDOR dephasing time is used, only backbone 13C signals are observed. The resulting spectra are used to determine spectral linewidths and to assess feasibility of assignment of uniformly labeled peptide.

  20. The small GTPase MoYpt7 is required for membrane fusion in autophagy and pathogenicity of Magnaporthe oryzae.

    PubMed

    Liu, Xiao-Hong; Chen, Si-Miao; Gao, Hui-Min; Ning, Guo-Ao; Shi, Huan-Bin; Wang, Yao; Dong, Bo; Qi, Yao-Yao; Zhang, Dong-Mei; Lu, Guo-Dong; Wang, Zong-Hua; Zhou, Jie; Lin, Fu-Cheng

    2015-11-01

    Rab GTPases are required for vesicle-vacuolar fusion during vacuolar biogenesis in fungi. To date, little is known about the biological functions of the Rab small GTPase components in Magnaporthe oryzae. In this study, we investigated MoYpt7 of M. oryzae, a homologue of the small Ras-like GTPase Ypt7 in Saccharomyces cerevisiae. Cellular localization assays showed that MoYpt7 was predominantly localized to vacuolar membranes. Using a targeted gene disruption strategy, a ΔMoYPT7 mutant was generated that exhibited defects in mycelial growth and production of conidia. The conidia of the ΔMoYPT7 mutant were malformed and defective in the formation of appressoria. Consequently, the ΔMoYPT7 mutant failed to cause disease in rice and barley. Furthermore, the ΔMoYPT7 mutant showed impairment in autophagy, breached cell wall integrity, and higher sensitivity to both calcium and heavy metal stress. Transformants constitutively expressing an active MoYPT7 allele (MoYPT7-CA, Gln67Leu) exhibited distinct phenotypes from the ΔMoYPT7 mutant. Expression of MoYPT7-CA in MoYpt7 reduced pathogenicity and produced more appressoria-forming single-septum conidia. These results indicate that MoYPT7 is required for fungal morphogenesis, vacuole fusion, autophagy, stress resistance and pathogenicity in M. oryzae.

  1. Characterization of the plasma membrane localization and orientation of HPV16 E5 for cell-cell fusion

    SciTech Connect

    Hu Lulin; Ceresa, Brian P.

    2009-10-10

    Human papillomavirus (HPV) is a non-enveloped DNA virus with an approx 8000 base pair genome. Infection with certain types of HPV is associated with cervical cancer, although the molecular mechanism by which HPV induces carcinogenesis is poorly understood. Three genes encoded by HPV16 are regarded as oncogenic - E5, E6, and E7. The role of E5 has been controversial. Expression of HPV16 E5 causes cell-cell fusion, an event that can lead to increased chromosomal instability, particularly in the presence of cell cycle checkpoint inhibitors like HPV16 E6 and E7. Using biochemical and cell biological assays to better understand HPV16 E5, we find that HPV16 E5 localizes to the plasma membrane with an intracellular amino terminus and an extracellular carboxyl-terminus. Further, HPV16 E5 must be expressed on both cells for cell fusion to occur. When the extracellular epitope of HPV16 E5 is targeted with an antibody, the number of bi-nucleated cells decreases.

  2. Herpes simplex virus 1 glycoprotein M and the membrane-associated protein UL11 are required for virus-induced cell fusion and efficient virus entry.

    PubMed

    Kim, In-Joong; Chouljenko, Vladimir N; Walker, Jason D; Kousoulas, Konstantin G

    2013-07-01

    Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.

  3. Phosphorylation of the C Terminus of RHD3 Has a Critical Role in Homotypic ER Membrane Fusion in Arabidopsis1[OPEN

    PubMed Central

    Ueda, Haruko; Yokota, Etsuo; Kuwata, Keiko; Kutsuna, Natsumaro; Mano, Shoji; Shimada, Tomoo; Tamura, Kentaro; Fukao, Yoichiro; Brandizzi, Federica; Shimmen, Teruo; Nishimura, Mikio

    2016-01-01

    The endoplasmic reticulum (ER) consists of dynamically changing tubules and cisternae. In animals and yeast, homotypic ER membrane fusion is mediated by fusogens (atlastin and Sey1p, respectively) that are membrane-associated dynamin-like GTPases. In Arabidopsis (Arabidopsis thaliana), another dynamin-like GTPase, ROOT HAIR DEFECTIVE3 (RHD3), has been proposed as an ER membrane fusogen, but direct evidence is lacking. Here, we show that RHD3 has an ER membrane fusion activity that is enhanced by phosphorylation of its C terminus. The ER network was RHD3-dependently reconstituted from the cytosol and microsome fraction of tobacco (Nicotiana tabacum) cultured cells by exogenously adding GTP, ATP, and F-actin. We next established an in vitro assay system of ER tubule formation with Arabidopsis ER vesicles, in which addition of GTP caused ER sac formation from the ER vesicles. Subsequent application of a shearing force to this system triggered the formation of tubules from the ER sacs in an RHD-dependent manner. Unexpectedly, in the absence of a shearing force, Ser/Thr kinase treatment triggered RHD3-dependent tubule formation. Mass spectrometry showed that RHD3 was phosphorylated at multiple Ser and Thr residues in the C terminus. An antibody against the RHD3 C-terminal peptide abolished kinase-triggered tubule formation. When the Ser cluster was deleted or when the Ser residues were replaced with Ala residues, kinase treatment had no effect on tubule formation. Kinase treatment induced the oligomerization of RHD3. Neither phosphorylation-dependent modulation of membrane fusion nor oligomerization has been reported for atlastin or Sey1p. Taken together, we propose that phosphorylation-stimulated oligomerization of RHD3 enhances ER membrane fusion to form the ER network. PMID:26684656

  4. Is the enthalpy of fusion of tris(acetylacetonato)metal(III) complexes affected by their potential energy in the crystal state?

    PubMed

    Sabolović, Jasmina; Mrak, Zeljko; Kostrun, Sanja; Janeković, August

    2004-12-27

    In this Article we present enthalpies of fusion and melting points obtained from new thermochemical measurements of tris(acetylacetonato)metal(III), M(acac)(3), complexes (M = Fe, Al, Cr, Mn, Co) using differential scanning calorimetry (DSC) and evaluate them in relation to their different values found in the literature. An enthalpy of fusion of 27.67 kJ mol(-)(1) was derived for Mn(acac)(3) from a symmetrical DSC thermogram captured for the first time. The enthalpy value was indirectly confirmed with the solubility measurements of Mn(acac)(3) in acetylacetone. A hypothesis has been stated that the enthalpy of fusion and the potential energy of M(acac)(3) in the crystal state may be related. To calculate molecular in-crystal potential energy, in this Article we proposed a molecular mechanics model for the M(acac)(3) class of compounds. Nine X-ray crystal structures of M(acac)(3) complexes (M = Fe, Al, V, Mn, Co, Cr, Sc) were included in the modeling. The conformational potential energy was minimized for a molecule surrounded by other molecules in the crystal lattice. The partial charges from two schemes, the electrostatic potential (ESP) fit and the natural population analysis (NPA), were used to construct two types of force fields to examine which force field type would yield a better fit with the experimental thermal properties. The final force fields were named FF-ESP and FF-NPA. Both force field sets reproduced well the experimental crystal data of nine M(acac)(3) complexes as well as of tris(3-methyl-2,4-pentanedionato-O,O')cobalt(III). Only in-crystal potential energies derived by FF-NPA yielded a significant correlation (correlation coefficient R = -0.71) with the measured enthalpies of fusion. The enthalpy of fusion for Co(acac)(3) could not be determined experimentally because of simultaneous decomposition and fusion, and it is predicted to be 33.2 kJ mol(-)(1) from the correlation regression line.

  5. A minimal phycobilisome: fusion and chromophorylation of the truncated core-membrane linker and phycocyanin.

    PubMed

    Tang, Kun; Zeng, Xiao-Li; Yang, Yi; Wang, Zhi-Bin; Wu, Xian-Jun; Zhou, Ming; Noy, Dror; Scheer, Hugo; Zhao, Kai-Hong

    2012-07-01

    Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli, the fused phycobiliproteins absorbed light in the range of 610-660nm, and fluoresced at ~670nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540-575nm, and fluorescing at ~585nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly. PMID:22465853

  6. A minimal phycobilisome: fusion and chromophorylation of the truncated core-membrane linker and phycocyanin.

    PubMed

    Tang, Kun; Zeng, Xiao-Li; Yang, Yi; Wang, Zhi-Bin; Wu, Xian-Jun; Zhou, Ming; Noy, Dror; Scheer, Hugo; Zhao, Kai-Hong

    2012-07-01

    Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli, the fused phycobiliproteins absorbed light in the range of 610-660nm, and fluoresced at ~670nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540-575nm, and fluorescing at ~585nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly.

  7. Identification and immunogenic potential of B cell epitopes of outer membrane protein OmpF of Aeromonas hydrophila in translational fusion with a carrier protein.

    PubMed

    Sharma, Mahima; Dixit, Aparna

    2015-08-01

    Aeromonas hydrophila, a ubiquitous and virulent bacterial pathogen, affects a variety of fishes, including Labeo rohita. Existing treatment strategies comprise antibiotic therapies and attenuated bacterial strain-based vaccines. No functional subunit vaccine has been available until now. Given their key role in determining pathogenicity, outer membrane proteins have been successfully explored as potential vaccine candidates. We have devised a direct strategy for eliminating non-specific responses by selectively aiming the immune response against specific immunodominant epitopes of the outer membrane protein F (OmpF) of A. hydrophila (AhOmpF). Five putative epitopes of AhOmpF predicted in silico were genetically conjugated with heat labile enterotoxin chain B of E. coli (LTB). Recombinant fusion proteins expressed in E. coli were purified from solubilized inclusion bodies and refolded. The fusion protein retained GM1 ganglioside receptor binding activity of LTB, indicating proper folding. Four of the five fusion proteins were found to be highly immunogenic. Of the four proteins, antisera against the fusion protein (anti-rEpiF1) harboring 66-80 amino acid residues of the OmpF gave maximum cross-reactivity with the targeted rOmpF in enzyme-linked immunosorbent assay (ELISA) and was able to recognize both fusion partners-rOmpF and rLTB-in Western blot. Antibody isotyping of the antisera and cytokine array analysis of the culture supernatants of splenocytes from sensitized mice manifested a mixed Th1/Th2 immune response with a bias toward Th2. Anti-rEpiF1 antibodies were able to bind to the cell membrane of live A. hydrophila cells and agglutinate them. Our results thus suggest that the OmpF epitope (66-80) in fusion with a carrier protein is a promising vaccine candidate against A. hydrophila.

  8. Construction and evaluation of PVC and sol-gel sensor membranes based on Mn(III)TPP-Cl. Application to valproate determination in pharmaceutical preparations.

    PubMed

    Santos, Emília M G; Araújo, Alberto N; Couto, Cristina M C M; Montenegro, M Conceição B S M

    2006-02-01

    The construction and general performance of new valproate-selective electrodes based on manganese(III) tetraphenylporphyrin [Mn(III)TPP-Cl], as an ionophore, are presented. The ionophore was incorporated into PVC and ceramic membranes (sol-gel) based on methyltriethoxysilane. The influence of membrane composition and pH and the effect of lipophilic cationic and anionic additives in PVC membranes were investigated concerning their influence on the slope, response time, selectivity and lifetime of the electrodes. The PVC membrane without additive and the sol-gel membrane presented slopes and practical limits of detection of -60.8 mV dec(-1) and 5x10(-6) mol l(-1) and -60.3 mV dec(-1) and 1x10(-4) mol l(-1), respectively. The sol-gel membranes displayed higher selectivity for valproate when compared with PVC membranes. These two types of electrodes were coupled to a sequential-injection analysis (SIA) system for the direct determination of valproate in pharmaceutical formulations. The association of Mn(III)TPP-Cl with the sol-gel support inserted in a SIA system provided potentiometric sensors with an analytical range of 1x10(-3)-5x10(-2) mol l(-1), with a sample rate of 55 samples per hour and a sample and carrier consumption of 140 and 2,500 microl per determination, respectively.

  9. Charged Multivesicular Body Protein 2B (CHMP2B) of the Endosomal Sorting Complex Required for Transport-III (ESCRT-III) Polymerizes into Helical Structures Deforming the Plasma Membrane*

    PubMed Central

    Bodon, Gilles; Chassefeyre, Romain; Pernet-Gallay, Karin; Martinelli, Nicolas; Effantin, Grégory; Hulsik, David Lutje; Belly, Agnès; Goldberg, Yves; Chatellard-Causse, Christine; Blot, Béatrice; Schoehn, Guy; Weissenhorn, Winfried; Sadoul, Rémy

    2011-01-01

    The endosomal sorting complexes required for transport (ESCRT-0-III) allow membrane budding and fission away from the cytosol. This machinery is used during multivesicular endosome biogenesis, cytokinesis, and budding of some enveloped viruses. Membrane fission is catalyzed by ESCRT-III complexes made of polymers of charged multivesicular body proteins (CHMPs) and by the AAA-type ATPase VPS4. How and which of the ESCRT-III subunits sustain membrane fission from the cytoplasmic surface remain uncertain. In vitro, CHMP2 and CHMP3 recombinant proteins polymerize into tubular helical structures, which were hypothesized to drive vesicle fission. However, this model awaits the demonstration that such structures exist and can deform membranes in cellulo. Here, we show that depletion of VPS4 induces specific accumulation of endogenous CHMP2B at the plasma membrane. Unlike other CHMPs, overexpressed full-length CHMP2B polymerizes into long, rigid tubes that protrude out of the cell. CHMP4s relocalize at the base of the tubes, the formation of which depends on VPS4. Cryo-EM of the CHMP2B membrane tubes demonstrates that CHMP2B polymerizes into a tightly packed helical lattice, in close association with the inner leaflet of the membrane tube. This association is tight enough to deform the lipid bilayer in cases where the tubular CHMP2B helix varies in diameter or is closed by domes. Thus, our observation that CHMP2B polymerization scaffolds membranes in vivo represents a first step toward demonstrating its structural role during outward membrane deformation. PMID:21926173

  10. A single amino acid substitution modulates low-pH-triggered membrane fusion of GP64 protein in Autographa californica and Bombyx mori nucleopolyhedroviruses

    SciTech Connect

    Katou, Yasuhiro; Yamada, Hayato; Ikeda, Motoko; Kobayashi, Michihiro

    2010-09-01

    We have previously shown that budded viruses of Bombyx mori nucleopolyhedrovirus (BmNPV) enter the cell cytoplasm but do not migrate into the nuclei of non-permissive Sf9 cells that support a high titer of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) multiplication. Here we show, using the syncytium formation assay, that low-pH-triggered membrane fusion of BmNPV GP64 protein (Bm-GP64) is significantly lower than that of AcMNPV GP64 protein (Ac-GP64). Mutational analyses of GP64 proteins revealed that a single amino acid substitution between Ac-GP64 H155 and Bm-GP64 Y153 can have significant positive or negative effects on membrane fusion activity. Studies using bacmid-based GP64 recombinant AcMNPV harboring point-mutated ac-gp64 and bm-gp64 genes showed that Ac-GP64 H155Y and Bm-GP64 Y153H substitutions decreased and increased, respectively, the multiplication and cell-to-cell spread of progeny viruses. These results indicate that Ac-GP64 H155 facilitates the low-pH-triggered membrane fusion reaction between virus envelopes and endosomal membranes.

  11. Determination of physical membrane properties of plant cell protoplasts via the electrofusion technique: prediction of optimal fusion yields and protoplast viability.

    PubMed

    Mehrle, W; Naton, B; Hampp, R

    1990-04-01

    By variation of physical parameters (field strength, pulse duration) which result in electrofusion and electroporation, properties of the plasma membrane of different types of plant cell protoplasts were analyzed. The lower threshold for that field pulse intensity at which membrane breakdown occurred (recorded as fusion event) depended on pulse duration, protoplast size, and protoplast type (tobacco, oat; vacuolated, evacuolated). This fusion characteristic of plant protoplasts can also be taken as a measure of the charging process of the membrane and allows thus a non-invasive determination of the time constant and the specific membrane capacitance. Although the fusion yield was comparable at pulse duration/field strength couples of, e.g., 10 μs/1.5 kV*cm(-1) and 200 μs/0.5 kV*cm(-1), hybrid viability was not. Rates of cell wall regeneration and cell division of tobacco mesophyll protoplasts were not affected but may have been increased at short pulse duration/high field strength. Plating efficiency, in contrast, was significantly decreased with longer pulse duration at low field strengths. PMID:24232787

  12. The yeast ATP-binding cassette (ABC) transporter Ycf1p enhances the recruitment of the soluble SNARE Vam7p to vacuoles for efficient membrane fusion.

    PubMed

    Sasser, Terry L; Lawrence, Gus; Karunakaran, Surya; Brown, Christopher; Fratti, Rutilio A

    2013-06-21

    The Saccharomyces cerevisiae vacuole contains five ATP-binding cassette class C (ABCC) transporters, including Ycf1p, a family member that was originally characterized as a Cd(2+) transporter. Ycf1p has also been found to physically interact with a wide array of proteins, including factors that regulate vacuole homeostasis. In this study, we examined the role of Ycf1p and other ABCC transporters in the regulation of vacuole homotypic fusion. We found that deletion of YCF1 attenuated in vitro vacuole fusion by up to 40% relative to wild-type vacuoles. Plasmid-expressed wild-type Ycf1p rescued the deletion phenotype; however, Ycf1p containing a mutation of the conserved Lys-669 to Met in the Walker A box of the first nucleotide-binding domain (Ycf1p(K669M)) was unable to complement the fusion defect of ycf1Δ vacuoles. This indicates that the ATPase activity of Ycf1p is required for its function in regulating fusion. In addition, we found that deleting YCF1 caused a striking decrease in vacuolar levels of the soluble SNARE Vam7p, whereas total cellular levels were not altered. The attenuated fusion of ycf1Δ vacuoles was rescued by the addition of recombinant Vam7p to in vitro experiments. Thus, Ycf1p contributes in the recruitment of Vam7p to the vacuole for efficient membrane fusion.

  13. A recombinant fusion protein containing the domain III of the dengue-2 envelope protein is immunogenic and protective in nonhuman primates.

    PubMed

    Hermida, Lisset; Bernardo, Lídice; Martín, Jorge; Alvarez, Mayling; Prado, Irina; López, Carlos; Sierra, Beatriz de la C; Martínez, Rafael; Rodríguez, Rosmary; Zulueta, Aída; Pérez, Ana B; Lazo, Laura; Rosario, Delfina; Guillén, Gerardo; Guzmán, María G

    2006-04-12

    We have previously reported the construction and evaluation in mice of recombinant fusion proteins formed by a fragment (aa 286-426) of the dengue envelope protein and the P64k protein from Neisseria meningitidis. In this work we describe the immunization of Macaca fascicularis monkeys with two variants of these proteins [PD3 (insertion variant) and PD5 (fusion variant)] corresponding to serotype 2. Four doses of the proteins adjuvated in Freund's adjuvant were administered and the kinetics of antibody induction was monitored by ELISA and neutralization tests. Monkeys receiving PD3 or PD5 developed functional antibodies (Abs) in a dose-dependent manner. Following challenge with 5 log PFU of wild type dengue-2 virus (DEN2), animals immunized with PD5 were protected from developing viremia. These results constitute a proof-of-concept demonstrating that a fragment of the dengue envelope protein, containing the domain III and produced as a recombinant fusion protein in Escherichia coli, induces functional and protective immunity in a nonhuman primate model.

  14. Versatile cellular uptake mediated by catanionic vesicles: simultaneous spontaneous membrane fusion and endocytosis.

    PubMed

    Mauroy, Chloé; Castagnos, Pauline; Orio, Julie; Blache, Marie-Claire; Rico-Lattes, Isabelle; Teissié, Justin; Rols, Marie-Pierre; Blanzat, Muriel

    2015-01-01

    Lactose-derived catanionic vesicles offer unique opportunities to overcome cellular barriers. These potential nanovectors, very easy to formulate as drug delivery systems, are able to encapsulate drugs of various hydrophilicity. This article highlights versatile interaction mechanisms between these catanionic vesicles, labeled with hydrophilic and amphiphilic fluorescent probes, and a mammalian cell line, Chinese Hamster Ovary. Confocal microscopy and flow cytometry techniques show that these vesicles are internalized by cells through cellular energy dependent processes, as endocytosis, but are simultaneously able to spontaneously fuse with cell plasma membranes and release their hydrophilic content directly inside the cytosol. Such innovative and polyvalent nanovectors, able to deliver their content via different internalization pathways, would positively be a great progress for the coadministration of drugs of complementary efficiency. PMID:25310849

  15. SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors

    PubMed Central

    Mojica, Sergio A.; Hovis, Kelley M.; Frieman, Matthew B.; Tran, Bao; Hsia, Ru-ching; Ravel, Jacques; Jenkins-Houk, Clifton; Wilson, Katherine L.; Bavoil, Patrik M.

    2015-01-01

    SINC, a new type III secreted protein of the avian and human pathogen Chlamydia psittaci, uniquely targets the nuclear envelope of C. psittaci–infected cells and uninfected neighboring cells. Digitonin-permeabilization studies of SINC-GFP–transfected HeLa cells indicate that SINC targets the inner nuclear membrane. SINC localization at the nuclear envelope was blocked by importazole, confirming SINC import into the nucleus. Candidate partners were identified by proximity to biotin ligase-fused SINC in HEK293 cells and mass spectrometry (BioID). This strategy identified 22 candidates with high confidence, including the nucleoporin ELYS, lamin B1, and four proteins (emerin, MAN1, LAP1, and LBR) of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, chromatin organization, and gene silencing. GFP-SINC association with the native LEM-domain protein emerin, a conserved component of nuclear “lamina” structure, or with a complex containing emerin was confirmed by GFP pull down. Our findings identify SINC as a novel bacterial protein that targets the nuclear envelope with the capability of globally altering nuclear envelope functions in the infected host cell and neighboring uninfected cells. These properties may contribute to the aggressive virulence of C. psittaci. PMID:25788290

  16. Chlamydia psittaci comparative genomics reveals intraspecies variations in the putative outer membrane and type III secretion system genes

    PubMed Central

    Wolff, Bernard J.; Morrison, Shatavia S.; Pesti, Denise; Ganakammal, Satishkumar Ranganathan; Srinivasamoorthy, Ganesh; Changayil, Shankar; Weil, M. Ryan; MacCannell, Duncan; Rowe, Lori; Frace, Michael; Ritchie, Branson W.; Dean, Deborah

    2015-01-01

    Chlamydia psittaci is an obligate intracellular bacterium that can cause significant disease among a broad range of hosts. In humans, this organism may cause psittacosis, a respiratory disease that can spread to involve multiple organs, and in rare untreated cases may be fatal. There are ten known genotypes based on sequencing the major outer-membrane protein gene, ompA, of C. psittaci. Each genotype has overlapping host preferences and virulence characteristics. Recent studies have compared C. psittaci among other members of the Chlamydiaceae family and showed that this species frequently switches hosts and has undergone multiple genomic rearrangements. In this study, we sequenced five genomes of C. psittaci strains representing four genotypes, A, B, D and E. Due to the known association of the type III secretion system (T3SS) and polymorphic outer-membrane proteins (Pmps) with host tropism and virulence potential, we performed a comparative analysis of these elements among these five strains along with a representative genome from each of the remaining six genotypes previously sequenced. We found significant genetic variation in the Pmps and tbl3SS genes that may partially explain differences noted in C. psittaci host infection and disease. PMID:25887617

  17. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    SciTech Connect

    McBride, Corrin E.; Machamer, Carolyn E.

    2010-09-15

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

  18. A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.

    PubMed

    Esposito, Anthony M; Cheung, Pamela; Swartz, Talia H; Li, Hongru; Tsibane, Tshidi; Durham, Natasha D; Basler, Christopher F; Felsenfeld, Dan P; Chen, Benjamin K

    2016-03-01

    Enveloped virus entry occurs when viral and cellular membranes fuse releasing particle contents into the target cell. Human immunodeficiency virus (HIV) entry occurs by cell-free virus or virus transferred between infected and uninfected cells through structures called virological synapses. We developed a high-throughput cell-based assay to identify small molecule inhibitors of cell-free or virological synapse-mediated entry. An HIV clone carrying Cre recombinase as a Gag-internal gene fusion releases active Cre into cells upon viral entry activating a recombinatorial gene switch changing dsRed to GFP-expression. A screen of a 1998 known-biological profile small molecule library identified pharmacological HIV entry inhibitors that block both cell-free and cell-to-cell infection. Many top hits were noted as HIV inhibitors in prior studies, but not previously recognized as entry antagonists. Modest therapeutic indices for simvastatin and nigericin were observed in confirmatory HIV infection assays. This robust assay is adaptable to study HIV and heterologous viral pseudotypes.

  19. Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes

    SciTech Connect

    Yasuzaki, Yukari; Yamada, Yuma; Harashima, Hideyoshi

    2010-06-25

    Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we provide a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.

  20. Extraction of Gold(III) from Hydrochloric Acid Solutions with a PVC-based Polymer Inclusion Membrane (PIM) Containing Cyphos® IL 104

    PubMed Central

    Bonggotgetsakul, Ya Ya Nutchapurida; Cattrall, Robert W.; Kolev, Spas D.

    2015-01-01

    Abstract: Poly(vinyl chloride) (PVC) based polymer inclusion membranes (PIMs), with different concentrations of Cyphos® IL 104 as the membrane extractant/carrier, were studied for their ability to extract Au(III) from hydrochloric acid solutions. Some of the PIMs also contained one of the following plasticizers or modifiers: 2-nitrophenyloctyl ether, dioctylphthalate, 1-dodecanol, 1-tetradecanol, or tri(2-ethylhexyl) phosphate. The best performance, in terms of extraction rate and amount of Au(III) extracted, was exhibited by a PIM consisting of 25 wt% Cyphos® IL 104, 5 wt% 1-dodecanol, and 70 wt% PVC. An almost complete back-extraction of the Au(III) extracted from this membrane was achieved by using a 0.10 mol L−1 Na2SO3 receiver solution at pH 8. The stoichiometry of the extracted Au(III)/Cyphos® IL 104 adduct was determined as [P]+ [AuCl4]− H+ [PO2]− where [P]+ and [PO2]− represent trihexyl(tetradecyl) phosphonium and bis(2,4,4-trimethylpentyl) phosphinate ions, respectively. Back-extraction of Au(III) is suggested to occur by reduction of Au(III) to Au(I), with the formation of the species [Au(SO3)2]3− in the aqueous receiver solution. Loss of 1-dodecanol from the newly developed PIM to the aqueous solutions in contact with it was observed, which indicated that this membrane was suitable for single use in the efficient recovery of Au(III) from hydrochloric acid solutions of electronic scrap or recycled jewelry. PMID:26670259

  1. Extraction of Gold(III) from Hydrochloric Acid Solutions with a PVC-based Polymer Inclusion Membrane (PIM) Containing Cyphos(®) IL 104.

    PubMed

    Bonggotgetsakul, Ya Ya Nutchapurida; Cattrall, Robert W; Kolev, Spas D

    2015-12-08

    Poly(vinyl chloride) (PVC) based polymer inclusion membranes (PIMs), with different concentrations of Cyphos® IL 104 as the membrane extractant/carrier, were studied for their ability to extract Au(III) from hydrochloric acid solutions. Some of the PIMs also contained one of the following plasticizers or modifiers: 2-nitrophenyloctyl ether, dioctylphthalate, 1-dodecanol, 1-tetradecanol, or tri(2-ethylhexyl) phosphate. The best performance, in terms of extraction rate and amount of Au(III) extracted, was exhibited by a PIM consisting of 25 wt% Cyphos(®) IL 104, 5 wt% 1-dodecanol, and 70 wt% PVC. An almost complete back-extraction of the Au(III) extracted from this membrane was achieved by using a 0.10 mol L(-1) Na₂SO₃ receiver solution at pH 8. The stoichiometry of the extracted Au(III)/Cyphos® IL 104 adduct was determined as [P]⁺ [AuCl₄](-) H⁺ [PO₂](-) where [P]⁺ and [PO₂](-) represent trihexyl(tetradecyl) phosphonium and bis(2,4,4-trimethylpentyl) phosphinate ions, respectively. Back-extraction of Au(III) is suggested to occur by reduction of Au(III) to Au(I), with the formation of the species [Au(SO₃)₂](3-) in the aqueous receiver solution. Loss of 1-dodecanol from the newly developed PIM to the aqueous solutions in contact with it was observed, which indicated that this membrane was suitable for single use in the efficient recovery of Au(III) from hydrochloric acid solutions of electronic scrap or recycled jewelry.

  2. Transmitter induced calcium entry across the post-synaptic membrane at frog end-plates measured using arsenazo III.

    PubMed

    Miledi, R; Parker, I; Schalow, G

    1980-03-01

    1. The Ca2+ influx occurring across the post-synaptic membrane during transmitter action was studied at the frog neuromuscular junction, using the Ca sensitive dye arsenazo III to monitor the resulting changes in free myoplasmic Ca2+ concentration. 2. Calibration experiments showed a linear relationship between the amount of Ca2+ injected by ionophoresis into a muscle fibre, and the peak size of the arsenazo light absorbance record. 3. Ionophoretic application of acetylcholine (ACh) to voltage clamped end-plates gave rise to an arsenazo signal. The size of this response varied with the Ca2+ concentration in the bathing solution. 4. The arsenazo light response increased in size steeply, and non-linearly, with hyperpolarization of the end-plate membrane, even when the end-plate current increased approximately linearly with hyperpolarization. The voltage dependence of the light response could be fitted well by an exponential with a voltage constant of 28 mV. Changes in Ca2+ concentration of the bathing medium had little effect on this relationship. 5. At end-plates bathed in isotonic CaCl2 solution the voltage dependence of both the arsenazo light response, and the end-plate current showed a closely similar, non-linear relationship. 6. Addition of 12 mM-Co2+ to a bathing solution initially containing 12 mM-Ca2+ substantially reduced the size of the arsenazo light response, and the voltage dependence of this response became more linear. 7. Arsenazo light responses were also recorded in response to transmitter release evoked by nerve stimulation. The size of the nerve evoked light response showed a non-linear voltage dependence, whilst the end-plate current was a linear function of membrane potential.

  3. Herpes simplex virus glycoproteins gB and gH function in fusion between the virion envelope and the outer nuclear membrane.

    PubMed

    Farnsworth, Aaron; Wisner, Todd W; Webb, Michael; Roller, Richard; Cohen, Gary; Eisenberg, Roselyn; Johnson, David C

    2007-06-12

    Herpesviruses must traverse the nuclear envelope to gain access to the cytoplasm and, ultimately, to exit cells. It is believed that herpesvirus nucleocapsids enter the perinuclear space by budding through the inner nuclear membrane (NM). To reach the cytoplasm these enveloped particles must fuse with the outer NM and the unenveloped capsids then acquire a second envelope in the trans-Golgi network. Little is known about the process by which herpesviruses virions fuse with the outer NM. Here we show that a herpes simplex virus (HSV) mutant lacking both the two putative fusion glycoproteins gB and gH failed to cross the nuclear envelope. Enveloped virions accumulated in the perinuclear space or in membrane vesicles that bulged into the nucleoplasm (herniations). By contrast, mutants lacking just gB or gH showed only minor or no defects in nuclear egress. We concluded that either HSV gB or gH can promote fusion between the virion envelope and the outer NM. It is noteworthy that fusion associated with HSV entry requires the cooperative action of both gB and gH, suggesting that the two types of fusion (egress versus entry) are dissimilar processes.

  4. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion.

    PubMed

    Madu, Ikenna G; Belouzard, Sandrine; Whittaker, Gary R

    2009-10-25

    The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

  5. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

    PubMed Central

    Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

    2013-01-01

    The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1–S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell–cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion. PMID:19717178

  6. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

    SciTech Connect

    Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

    2009-10-25

    The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

  7. FINESSE: study of the issues, experiments and facilities for fusion nuclear technology research and development. Interim report. Volume III

    SciTech Connect

    Abdou, M.

    1984-10-01

    This chapter deals with the analysis and engineering scaling of solid breeded blankets. The limits under which full component behavior can be achieved under changed test conditions are explored. The characterization of these test requirements for integrated testing contributes to the overall test matrix and test plan for the understanding and development of fusion nuclear technology. The second chapter covers the analysis and engineering scaling of liquid metal blankets. The testing goals for a complete blanket program are described. (MOW)

  8. TRAIL-Death Receptor 4 Signaling via Lysosome Fusion and Membrane Raft Clustering In Coronary Arterial Endothelial Cells: Evidence from ASM Knockout Mice

    PubMed Central

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M.; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2012-01-01

    Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) and its receptor death receptor 4 (DR4) have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation and leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MRs) clustering and formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and its co-localization with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1+/+) mice. Further, TRAIL triggered ASM translocation, ceramide production and NADPH oxidase aggregation in MR clusters in Smpd1+/+ CAECs, whereas these observations were not found in Smpd1−/− CAECs. Moreover, ASM deficiency reduced TRAIL-induced O2−· production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer (FRET), we found that Lamp-1 (lysosome membrane marker protein) and ganglioside GM1 (MR marker) were trafficking together in Smpd1+/+ CAECs, which was absent in Smpd1−/− CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1−/− CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking and fusion with membrane and formation of MR redox signaling platforms, which may

  9. MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

    PubMed

    Lin, Hong Ting; Bavro, Vassiliy N; Barrera, Nelson P; Frankish, Helen M; Velamakanni, Saroj; van Veen, Hendrik W; Robinson, Carol V; Borges-Walmsley, M Inês; Walmsley, Adrian R

    2009-01-01

    Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.

  10. MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.

    PubMed

    Lin, Hong Ting; Bavro, Vassiliy N; Barrera, Nelson P; Frankish, Helen M; Velamakanni, Saroj; van Veen, Hendrik W; Robinson, Carol V; Borges-Walmsley, M Inês; Walmsley, Adrian R

    2009-01-01

    Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled. PMID:18955484

  11. MacB ABC Transporter Is a Dimer Whose ATPase Activity and Macrolide-binding Capacity Are Regulated by the Membrane Fusion Protein MacA*S⃞

    PubMed Central

    Lin, Hong Ting; Bavro, Vassiliy N.; Barrera, Nelson P.; Frankish, Helen M.; Velamakanni, Saroj; van Veen, Hendrik W.; Robinson, Carol V.; Borges-Walmsley, M. Inês; Walmsley, Adrian R.

    2009-01-01

    Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled. PMID:18955484

  12. H1N1 Swine Influenza Viruses Differ from Avian Precursors by a Higher pH Optimum of Membrane Fusion

    PubMed Central

    Baumann, Jan; Kouassi, Nancy Mounogou; Foni, Emanuela; Klenk, Hans-Dieter

    2015-01-01

    ABSTRACT The H1N1 Eurasian avian-like swine (EAsw) influenza viruses originated from an avian H1N1 virus. To characterize potential changes in the membrane fusion activity of the hemagglutinin (HA) during avian-to-swine adaptation of the virus, we studied EAsw viruses isolated in the first years of their circulation in pigs and closely related contemporary H1N1 viruses of wild aquatic birds. Compared to the avian viruses, the swine viruses were less sensitive to neutralization by lysosomotropic agent NH4Cl in MDCK cells, had a higher pH optimum of hemolytic activity, and were less stable at acidic pH. Eight amino acid substitutions in the HA were found to separate the EAsw viruses from their putative avian precursor; four substitutions—T492S, N722D, R752K, and S1132F—were located in the structural regions of the HA2 subunit known to play a role in acid-induced conformational transition of the HA. We also studied low-pH-induced syncytium formation by cell-expressed HA proteins and found that the HAs of the 1918, 1957, 1968, and 2009 pandemic viruses required a lower pH for fusion induction than did the HA of a representative EAsw virus. Our data show that transmission of an avian H1N1 virus to pigs was accompanied by changes in conformational stability and fusion promotion activity of the HA. We conclude that distinctive host-determined fusion characteristics of the HA may represent a barrier for avian-to-swine and swine-to-human transmission of influenza viruses. IMPORTANCE Continuing cases of human infections with zoonotic influenza viruses highlight the necessity to understand which viral properties contribute to interspecies transmission. Efficient binding of the HA to cellular receptors in a new host species is known to be essential for the transmission. Less is known about required adaptive changes in the membrane fusion activity of the HA. Here we show that adaptation of an avian influenza virus to pigs in Europe in 1980s was accompanied by mutations in

  13. Membrane triangles with corner drilling freedoms. III - Implementation and performance evaluation

    NASA Technical Reports Server (NTRS)

    Felippa, Carlos A.; Alexander, Scott

    1992-01-01

    This paper completes a three-part series on the formulation of 3-node, 9-dof membrane triangles with corner drilling freedoms based on parametrized variational principles. The first four sections cover element implementation details including determination of optimal parameters and treatment of distributed loads. Then three elements of this type, labeled ALL, FF and EFF-ANDES, are tested on standard plane stress problems. ALL represents numerically integrated versions of Allman's 1988 triangle; FF is based on the free formulation triangle presented by Bergan and Felippa in 1985; and EFF-ANDES represent two different formulations of the optimal triangle derived in Parts I and II. The numerical studies indicate that the ALL, FF and EFF-ANDES elements are comparable in accuracy for elements of unitary aspect ratios. The ALL elements are found to stiffen rapidly in inplane bending for high aspect ratios, whereas the FF and EFF elements maintain accuracy. The EFF and ANDES implementations have a moderate edge in formation speed over the FF.

  14. Inhibition of a type III secretion system by the deletion of a short loop in one of its membrane proteins

    SciTech Connect

    Meshcheryakov, Vladimir A.; Kitao, Akio; Matsunami, Hideyuki; Samatey, Fadel A.

    2013-05-01

    Crystal structures of the cytoplasmic domain of FlhB from S. typhimurium and A. aeolicus were solved at 2.45 and 2.55 Å resolution, respectively. The deletion of a short loop in the cytoplasmic domain of Salmonella FlhB completely abolishes secretion by the type III secretion system. A molecular-dynamics simulation shows that the deletion of the loop affects the flexibility of a linker between the transmembrane and cytoplasmic domains of FlhB. The membrane protein FlhB is a highly conserved component of the flagellar secretion system. It is composed of an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhB{sub C}). Here, the crystal structures of FlhB{sub C} from Salmonella typhimurium and Aquifex aeolicus are described at 2.45 and 2.55 Å resolution, respectively. These flagellar FlhB{sub C} structures are similar to those of paralogues from the needle type III secretion system, with the major difference being in a linker that connects the transmembrane and cytoplasmic domains of FlhB. It was found that deletion of a short flexible loop in a globular part of Salmonella FlhB{sub C} leads to complete inhibition of secretion by the flagellar secretion system. Molecular-dynamics calculations demonstrate that the linker region is the most flexible part of FlhB{sub C} and that the deletion of the loop reduces this flexibility. These results are in good agreement with previous studies showing the importance of the linker in the function of FlhB and provide new insight into the relationship between the different parts of the FlhB{sub C} molecule.

  15. Micropipette manipulation technique for the monitoring of pH-dependent membrane lysis as induced by the fusion peptide of influenza virus.

    PubMed Central

    Soltesz, S A; Hammer, D A

    1995-01-01

    We have assembled a micropipette aspiration assay to measure membrane destabilization events in which large (20-30 microns diameter) unilamellar vesicles are manipulated and exposed to membrane destabilizing agents. Single events can be seen with a light microscope and are recorded using both a video camera and a photomultiplier tube. We have performed experiments with a wild-type fusion peptide from influenza virus (X31) and found that it induces pH-dependent, stochastic lysis of large unilamellar vesicles. The rate and extent of lysis are both maximum at pH 5; the maximum rate of lysis is 0.018 s-1 at pH 5. An analysis of our data indicates that the lysis is not correlated either to the size of the vesicles or to the tension created in the vesicle membranes by aspiration. Images FIGURE 1 FIGURE 2 FIGURE 3 FIGURE 4 PMID:7711257

  16. Rab3A is a new interacting partner of synaptotagmin I and may modulate synaptic membrane fusion through a competitive mechanism

    SciTech Connect

    Xie, Chunliang; Li, Jianglin; Guo, Tianyao; Yan, Yizhong; Tang, Cheng; Wang, Ying; Chen, Ping; Wang, Xianchun; Liang, Songping

    2014-02-21

    Highlights: • Rab3A has been found to be a novel interacting protein of synaptotagmin I. • Rab3A binds to synaptotagmin I in a Ca{sup 2+}-independent manner. • KKKK motif in C2B domain of synaptotagmin I is a key site for Rab3A binding. • Rab3A competitively inhibits the binding of C2B in synaptotagmin I to syntaxin 1B. • Rab3A may regulate synaptic membrane fusion and exocytosis in a competitive manner. - Abstract: Rab3 and synaptotagmin have been reported to be the key proteins that have opposite actions but cooperatively play critical regulatory roles in selecting and limiting the number of vesicles released at central synapses. However, the exact mechanism has not been fully understood. In this study, Rab3A and synaptotagmin I, the most abundant isoforms of Rab3 and synaptotagmin, respectively, in brain were for the first time demonstrated to directly interact with each other in a Ca{sup 2+}-independent manner, and the KKKK motif in the C2B domain of synaptotagmin I was a key site for the Rab3A binding, which was further confirmed by the competitive inhibition of inositol hexakisphosphate. Further studies demonstrated that Rab3A competitively affected the synaptotagmin I interaction with syntaxin 1B that was involved in membrane fusion during the synaptic vesicle exocytosis. These data indicate that Rab3A is a new synaptotagmin I interacting partner and may participate in the regulation of synaptic membrane fusion and thus the vesicle exocytosis by competitively modulating the interaction of synaptotagmin with syntaxin of the t-SNARE complex in presynaptic membranes.

  17. Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways

    PubMed Central

    Rival, Thomas; Macchi, Marc; Arnauné-Pelloquin, Laetitia; Poidevin, Mickael; Maillet, Frédéric; Richard, Fabrice; Fatmi, Ahmed; Belenguer, Pascale; Royet, Julien

    2011-01-01

    Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent. PMID:21274005

  18. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion.

    PubMed

    Paquette, Stéphane G; Banner, David; Chi, Le Thi Bao; Leόn, Alberto J; Xu, Luoling; Ran, Longsi; Huang, Stephen S H; Farooqui, Amber; Kelvin, David J; Kelvin, Alyson A

    2014-01-01

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus-epithelial cell interaction.

  19. Rapid purification of recombinant dengue and West Nile virus envelope Domain III proteins by metal affinity membrane chromatography.

    PubMed

    Tan, Lik Chern Melvin; Chua, Anthony Jin Shun; Goh, Li Shan Liza; Pua, Shu Min; Cheong, Yuen Kuen; Ng, Mah Lee

    2010-11-01

    Arthropod-borne flaviviruses such as dengue virus (DENV) and West Nile virus (WNV) pose significant health threats to the global community. Due to escalating numbers of DENV and WNV infections worldwide, development of an effective vaccine remains a global health priority. As flavivirus envelope Domain III (DIII) protein is highly immunogenic and capable of inducing neutralizing antibodies against wild-type virus, it is both a potential protein subunit vaccine candidate and a suitable diagnostic reagent. Here, we describe the use of metal affinity membrane chromatography as a rapid and improved alternative for the purification of recombinant DIII (rDIII) antigens from DENV serotypes 1-4 and WNV - New York, Sarafend, Wengler and Kunjin strains. Optimum conditions for the expression, solubilization, renaturation and purification of these proteins were established. The purified proteins were confirmed by MALDI-TOF mass spectrometry and ELISA using antibodies raised against the respective viruses. Biological function of the purified rDIII proteins was confirmed by their ability to generate DIII-specific antibodies in mice that could neutralize the virus.

  20. Effective As(III) Removal by A Multi-Charged Hydroacid Complex Draw Solute Facilitated Forward Osmosis-Membrane Distillation (FO-MD) Processes.

    PubMed

    Ge, Qingchun; Han, Gang; Chung, Tai-Shung

    2016-03-01

    Effective removal of As(III) from water by an oxalic acid complex with the formula of Na3[Cr(C2O4)3] (Na-Cr-OA) is demonstrated via an forward osmosis-membrane distillation (FO-MD) hybrid system in this study. Na-Cr-OA first proved its superiority as a draw solute with high water fluxes and negligible reverse fluxes in FO, then a systematic investigation of the Na-Cr-OA promoted FO process was conducted to ascertain the factors in As(III) removal. Relatively high water fluxes of 28 LMH under the FO mode and 74 LMH under the pressure retarded osmosis (PRO) mode were achieved when using a 1000 ppm As(III) solution as the feed and 1.0 M Na-Cr-OA as the draw solution at 60 °C. As(III) removal with a water recovery up to 21.6% (FO mode) and 48.3% (PRO mode) were also achieved in 2 h. An outstanding As(III) rejection with 30-3000 μg/L As(III) in the permeate was accomplished when As(III) feed solutions varied from 5 × 10(4) to 1 × 10(6) μg/L, superior to the best FO performance reported for As(III) removal. Incorporating MD into FO not only makes As(III) removal sustainable by reconcentrating the Na-Cr-OA solution simultaneously, but also reduces the As(III) concentration below 10 μg/L in the product water, meeting the WHO standard. PMID:26822310

  1. Effective As(III) Removal by A Multi-Charged Hydroacid Complex Draw Solute Facilitated Forward Osmosis-Membrane Distillation (FO-MD) Processes.

    PubMed

    Ge, Qingchun; Han, Gang; Chung, Tai-Shung

    2016-03-01

    Effective removal of As(III) from water by an oxalic acid complex with the formula of Na3[Cr(C2O4)3] (Na-Cr-OA) is demonstrated via an forward osmosis-membrane distillation (FO-MD) hybrid system in this study. Na-Cr-OA first proved its superiority as a draw solute with high water fluxes and negligible reverse fluxes in FO, then a systematic investigation of the Na-Cr-OA promoted FO process was conducted to ascertain the factors in As(III) removal. Relatively high water fluxes of 28 LMH under the FO mode and 74 LMH under the pressure retarded osmosis (PRO) mode were achieved when using a 1000 ppm As(III) solution as the feed and 1.0 M Na-Cr-OA as the draw solution at 60 °C. As(III) removal with a water recovery up to 21.6% (FO mode) and 48.3% (PRO mode) were also achieved in 2 h. An outstanding As(III) rejection with 30-3000 μg/L As(III) in the permeate was accomplished when As(III) feed solutions varied from 5 × 10(4) to 1 × 10(6) μg/L, superior to the best FO performance reported for As(III) removal. Incorporating MD into FO not only makes As(III) removal sustainable by reconcentrating the Na-Cr-OA solution simultaneously, but also reduces the As(III) concentration below 10 μg/L in the product water, meeting the WHO standard.

  2. Peptide and non-peptide HIV fusion inhibitors.

    PubMed

    Jiang, Shibo; Zhao, Qian; Debnath, Asim K

    2002-01-01

    Fusion of the HIV envelope with the target cell membrane is a critical step of HIV entry into the target cell. The HIV envelope glycoprotein gp41 plays an important role in the fusion of viral and target cell membranes and serves as an attractive target for development of HIV fusion inhibitors. The extracellular domain of gp41 contains three important functional regions, i.e. fusion peptide (FP), N- and C-terminal heptad repeats (NHR and CHR, respectively). The FP region is composed of hydrophobic, glycine-rich residues that are essential for the initial penetration of the target cell membrane. NHR and CHR regions consist of hydrophobic residues, which have the tendency to form alpha-helical coiled coils. During the process of fusion of HIV or HIV-infected cells with uninfected cells, FP inserts into the target cell membrane and subsequently the NHR and CHR regions change conformations and associate with each other to form a fusion-active gp41 core. Peptides derived from NHR and CHR regions, designated N- and C-peptides, respectively, have potent inhibitory activity against HIV fusion by binding to the CHR and NHR regions, respectively, to prevent the formation of the fusion-active gp41 core. C-peptide may also bind to FP, thereby blocking its insertion into the target cell membrane. One of the C-peptides, T-20, which is in the phase III clinical trials, has potent in vivo activity against HIV infection and is expected to become the first peptide HIV fusion inhibitory drug in the near future. However, this peptide HIV fusion inhibitor lacks oral availability and is sensitive to the proteolytic digestion. Therefore, it is essential to develop small molecular non-peptide HIV fusion inhibitors having a mechanism of action similar to the C-peptides. One of the approaches in identifying the inhibitors is to use an immunological assay to screen chemical libraries for the compounds that potentially block the interaction between the NHR and CHR regions to form a fusion

  3. Bacterial endosymbiont-derived lipopolysaccharides and a protein on symbiosome membranes in newly infected amoebae and their roles in lysosome-symbiosome fusion.

    PubMed

    Kim, K J; Na, Y E; Jeon, K W

    1994-01-01

    Experimental results are presented to support the view that symbiont-derived lipopolysaccharides are involved in the prevention of lysosome-symbiosome fusion in xD amoebae harboring bacterial endosymbionts. Monoclonal antibodies against lipopolysaccharides and a 96-kDa protein present on symbiosome membranes of amoebae were used to monitor the appearance of the membrane-specific components in newly infected amoebae with endosymbionts from xD amoebae. The lipopolysaccharides and protein appeared on the newly forming symbiosome membranes within 3 to 7 days, as detected by indirect immunofluorescence staining with monoclonal antibodies. The lysosome-symbiosome fusion was followed by double staining of two antigens with different monoclonal antibodies applied to the same amoeba. Antilipopolysaccharide monoclonal antibodies were detected by staining with a fluorescein isothiocyanate-conjugated secondary antibody, and a biotinylated anti-lysosomal protein monoclonal antibody was detected by staining with Texas Red-conjugated streptavidin. In xD amoebae injected with an antilipopolysaccharide antibody, lysosomes fused with some of the symbiosomes that did not fuse with lysosomes in noninjected cells. PMID:8262651

  4. Cdc42 and Actin Control Polarized Expression of TI-VAMP Vesicles to Neuronal Growth Cones and Their Fusion with the Plasma MembraneV⃞

    PubMed Central

    Alberts, Philipp; Rudge, Rachel; Irinopoulou, Theano; Danglot, Lydia; Gauthier-Rouvière, Cécile; Galli, Thierry

    2006-01-01

    Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth. PMID:16381811

  5. Cdc42 and actin control polarized expression of TI-VAMP vesicles to neuronal growth cones and their fusion with the plasma membrane.

    PubMed

    Alberts, Philipp; Rudge, Rachel; Irinopoulou, Theano; Danglot, Lydia; Gauthier-Rouvière, Cécile; Galli, Thierry

    2006-03-01

    Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP)-mediated fusion of intracellular vesicles with the plasma membrane is crucial for neurite outgrowth, a pathway not requiring synaptobrevin-dependent exocytosis. Yet, it is not known how the TI-VAMP membrane trafficking pathway is regulated or how it is coordinated with cytoskeletal dynamics within the growth cone that guide neurite outgrowth. Here, we demonstrate that TI-VAMP, but not synaptobrevin 2, concentrates in the peripheral, F-actin-rich region of the growth cones of hippocampal neurons in primary culture. Its accumulation correlates with and depends upon the presence of F-actin. Moreover, acute stimulation of actin remodeling by homophilic activation of the adhesion molecule L1 induces a site-directed, actin-dependent recruitment of the TI-VAMP compartment. Expression of a dominant-positive mutant of Cdc42, a key regulator of cell polarity, stimulates formation of F-actin- and TI-VAMP-rich filopodia outside the growth cone. Furthermore, we report that Cdc42 activates exocytosis of pHLuorin tagged TI-VAMP in an actin-dependent manner. Collectively, our data suggest that Cdc42 and regulated assembly of the F-actin network control the accumulation and exocytosis of TI-VAMP-containing membrane vesicles in growth cones to coordinate membrane trafficking and actin remodeling during neurite outgrowth.

  6. Biophysical Characterization of a Vaccine Candidate against HIV-1: The Transmembrane and Membrane Proximal Domains of HIV-1 gp41 as a Maltose Binding Protein Fusion

    PubMed Central

    Gong, Zhen; Martin-Garcia, Jose M.; Daskalova, Sasha M.; Craciunescu, Felicia M.; Song, Lusheng; Dörner, Katerina; Hansen, Debra T.; Yang, Jay-How; LaBaer, Joshua; Hogue, Brenda G.; Mor, Tsafrir S.; Fromme, Petra

    2015-01-01

    The membrane proximal region (MPR, residues 649–683) and transmembrane domain (TMD, residues 684–705) of the gp41 subunit of HIV-1’s envelope protein are highly conserved and are important in viral mucosal transmission, virus attachment and membrane fusion with target cells. Several structures of the trimeric membrane proximal external region (residues 662–683) of MPR have been reported at the atomic level; however, the atomic structure of the TMD still remains unknown. To elucidate the structure of both MPR and TMD, we expressed the region spanning both domains, MPR-TM (residues 649–705), in Escherichia coli as a fusion protein with maltose binding protein (MBP). MPR-TM was initially fused to the C-terminus of MBP via a 42 aa-long linker containing a TEV protease recognition site (MBP-linker-MPR-TM). Biophysical characterization indicated that the purified MBP-linker-MPR-TM protein was a monodisperse and stable candidate for crystallization. However, crystals of the MBP-linker-MPR-TM protein could not be obtained in extensive crystallization screens. It is possible that the 42 residue-long linker between MBP and MPR-TM was interfering with crystal formation. To test this hypothesis, the 42 residue-long linker was replaced with three alanine residues. The fusion protein, MBP-AAA-MPR-TM, was similarly purified and characterized. Significantly, both the MBP-linker-MPR-TM and MBP-AAA-MPR-TM proteins strongly interacted with broadly neutralizing monoclonal antibodies 2F5 and 4E10. With epitopes accessible to the broadly neutralizing antibodies, these MBP/MPR-TM recombinant proteins may be in immunologically relevant conformations that mimic a pre-hairpin intermediate of gp41. PMID:26295457

  7. Membrane fusion by an RGD-containing sequence from the core protein VP3 of hepatitis A virus and the RGA-analogue: implications for viral infection.

    PubMed

    Chávez, A; Pujol, M; Haro, I; Alsina, M A; Cajal, Y

    2001-01-01

    The interaction of an RGD-containing epitope from the hepatitis A virus VP3 capsid protein and its RGA-analogue with lipid membranes was studied by biophysical methods. Two types of model membrane were used: vesicles and monolayers spread at the air/water interface, with a composition that closely resembles the lipid moiety of hepatocyte membranes: PC/SM/PE/PC (40:33:12:15; PC: 1-palmitoyl-2-oleoylglycero-sn-3-phosphocholine; SM: sphingomyelin from chicken egg yolk; PE, 1,2-dipalmitoyl-phosphatidylethanolamine; PS: L-alpha-phosphatidyl-L-serine from bovine brain). In addition, zwitterionic PC/SM/PE (47:39:14) and cationic PC/SM/PE/DOTAP (40:33:12:15; DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane) membranes were also prepared in order to dissect the electrostatic and hydrophobic components in the interaction. Changes in tryptophan fluorescence, acrylamide quenching, and resonance energy transfer experiments in the presence of vesicles, as well as the kinetics of insertion in monolayers, indicate that both peptides bind to the three types of membrane at neutral and acidic pH; however, binding is irreversible only at low pH. Membrane-destabilizing and fusogenic activities are triggered by acidification at pH 4-6, characteristic of the endosome. Fluorescence experiments show that VP3-RGD and VP3-RGA induce mixing of lipids and leakage or mixing of aqueous contents in anionic and cationic vesicles at pH 4-6, indicating leaky fusion. Interaction with zwitterionic vesicles (PC/SM/PE) results in leakage without lipid mixing, indicating pore formation. Replacement of aspartic acid in the RGD motif by alanine maintains the membrane-destabilizing properties of the peptide at low pH, but not its antigenicity. Since the RGD tripeptide is related to receptor-mediated cell adhesion and antigenicity, results suggest that receptor binding is not a molecular requirement for fusion. The possible involvement of peptide-induced membrane destabilization in the mechanism of hepatitis A

  8. Membrane-surfactant interactions. The role of surfactant in mitochondrial complex III-phospholipid-Triton X-100 mixed micelles

    SciTech Connect

    Valpuesta, J.M.; Arrondo, J.L.; Barbero, M.C.; Pons, M.; Goni, F.M.

    1986-05-15

    Complex III (ubiquinol-cytochrome c reductase) was purified from beef heart mitochondria in the form of protein-phospholipid-Triton X-100 mixed micelles (about 1:80:100 molar ratio). Detergent may be totally removed by sucrose density gradient centrifugation, and the resulting lipoprotein complexes retain full enzyme activity. In order to understand the role of surfactant in the mixed micelles, and the interaction of Triton X-100 with integral membrane proteins and phospholipid bilayers, both the protein-lipid-surfactant mixed micelles and the detergent-free lipoprotein system were examined from the point of view of particle size and ultrastructure, enzyme activity, tryptophan fluorescence quenching, 31P NMR, and Fourier transform infrared spectroscopy. The NMR and IR spectroscopic studies show that surfactant withdrawal induces a profound change in phospholipid architecture, from a micellar to a lamellar-like phase. However, electron microscopic observations fail to reveal the existence of lipid bilayers in the absence of detergent. We suggest that, under these conditions, the lipid:protein molar ratio (80:1) is too low to permit the formation of lipid bilayer planes, but the relative orientation and mobility of phospholipids with respect to proteins is similar to that of the lamellar phase. Protein conformational changes are also detected as a consequence of surfactant removal. Fourier transform infrared spectroscopy indicates an increase of peptide beta-structure in the absence of Triton X-100; changes in the amide II/amide I intensity ratio are also detected, although the precise meaning of these observations is unclear.

  9. The Mxi-Spa Type III Secretory Pathway of Shigella flexneri Requires an Outer Membrane Lipoprotein, MxiM, for Invasin Translocation

    PubMed Central

    Schuch, Raymond; Maurelli, Anthony T.

    1999-01-01

    Invasion of epithelial cells by Shigella flexneri is mediated by a set of translocated bacterial invasins, the Ipa proteins, and its dedicated type III secretion system, called Mxi-Spa. We show here that mxiM, part of the mxi-spa locus in the S. flexneri virulence plasmid, encodes an indispensable type III secretion apparatus component, required for both Ipa translocation and tissue culture cell invasion. We demonstrated that mature MxiM, first identified as a putative lipoprotein, is lipidated in vivo. Consistent with features of known lipoproteins, MxiM (i) can be labeled with [3H]palmitate and [2-3H]glycerol, (ii) is associated with the cell envelope, (iii) is secreted independently of the type III pathway, and (iv) requires an intact lipoprotein modification and processing site for full activity. The lipidated form of MxiM was detected primarily in the outer membrane, where it establishes a peripheral association with the inner leaflet. Through analysis of subcellular Ipa distribution in a mxiM null mutant background, MxiM was found to be required for the assembly and/or function of outer, but not inner, membrane regions of Mxi-Spa. This function probably requires interactions with other Mxi-Spa subunits within the periplasmic space. We discuss implications of these findings with respect to the function of MxiM and the structure of Mxi-Spa as a whole. PMID:10085046

  10. Domains II and III of Bacillus thuringiensis Cry1Ab Toxin Remain Exposed to the Solvent after Insertion of Part of Domain I into the Membrane*

    PubMed Central

    Zavala, Luis Enrique; Pardo-López, Liliana; Cantón, Pablo Emiliano; Gómez, Isabel; Soberón, Mario; Bravo, Alejandra

    2011-01-01

    Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially for the control of economical important insect pests. These are pore-forming toxins that interact with different receptors in the insect gut, forming pores in the apical membrane causing cell burst and insect death. Elucidation of the structure of the membrane-inserted toxin is important to fully understand its mechanism of action. One hypothesis proposed that the hairpin of α-helices 4–5 of domain I inserts into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane surface in an umbrella-like conformation. However, a second hypothesis proposed that the three domains of the Cry toxin insert into the bilayer without major conformational changes. In this work we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and labeled them with different fluorescent probes that have different responses to solvent polarity. Different soluble quenchers as well as a membrane-bound quencher were used to compare the properties of the soluble and brush border membrane-inserted forms of Cry1Ab toxin. The fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin remain in the surface of the membrane and only a discrete region of domain I is inserted into the lipid bilayer, supporting the umbrella model of toxin insertion. PMID:21464133

  11. Domains II and III of Bacillus thuringiensis Cry1Ab toxin remain exposed to the solvent after insertion of part of domain I into the membrane.

    PubMed

    Zavala, Luis Enrique; Pardo-López, Liliana; Cantón, Pablo Emiliano; Gómez, Isabel; Soberón, Mario; Bravo, Alejandra

    2011-05-27

    Bacillus thuringiensis produces insecticidal proteins named Cry toxins, that are used commercially for the control of economical important insect pests. These are pore-forming toxins that interact with different receptors in the insect gut, forming pores in the apical membrane causing cell burst and insect death. Elucidation of the structure of the membrane-inserted toxin is important to fully understand its mechanism of action. One hypothesis proposed that the hairpin of α-helices 4-5 of domain I inserts into the phospholipid bilayer, whereas the rest of helices of domain I are spread on the membrane surface in an umbrella-like conformation. However, a second hypothesis proposed that the three domains of the Cry toxin insert into the bilayer without major conformational changes. In this work we constructed single Cys Cry1Ab mutants that remain active against Manduca sexta larvae and labeled them with different fluorescent probes that have different responses to solvent polarity. Different soluble quenchers as well as a membrane-bound quencher were used to compare the properties of the soluble and brush border membrane-inserted forms of Cry1Ab toxin. The fluorescence and quenching analysis presented here, revealed that domains II and III of the toxin remain in the surface of the membrane and only a discrete region of domain I is inserted into the lipid bilayer, supporting the umbrella model of toxin insertion.

  12. Disposable terbium (III) salicylate complex imprinted membrane using solid phase surface fluorescence method for fast separation and detection of salicylic acid in pharmaceuticals and human urine.

    PubMed

    Huang, Jianxiang; Hu, Yufei; Hu, Yuling; Li, Gongke

    2013-03-30

    In this work, a simple, low cost, selective and sensitive complex imprinted membrane (CIM) for solid-phase fluorescent detection was developed with terbium (III) salicylate as complex template. Terbium-sensitized luminescence was employed for monitoring salicylic acid (SA) based on the fluorescence enhancement effect of benzoic acid derivatives on lanthanide ion Tb (III). The resulting CIM showed good fluorescent response and high selectivity towards SA with Tb as pivot in protic solvents, while demonstrating better analytical performance than the controlled membranes. The optimized adsorption time was 10 min, indicating rapid kinetics of the imprinted membrane. The linear response of CIM to SA was from 0.20 to 10mg/L with limit of detection (LOD) of 0.040 mg/L. The prepared CIM was successfully applied to the analysis of salicylic acid in pharmaceuticals and spiked human urine with recoveries of 80.6%-88.1%. The analytical results of the proposed method were in good agreement with those obtained by high performance liquid chromatography (HPLC) method, indicating that the developed membrane has acceptable practicability for fast determination of SA in real samples.

  13. An emergency response team for membrane repair

    NASA Technical Reports Server (NTRS)

    McNeil, Paul L.; Kirchhausen, Tom

    2005-01-01

    On demand, rapid Ca(2+)-triggered homotypic and exocytic membrane-fusion events are required to repair a torn plasma membrane, and we propose that this emergency-based fusion differs fundamentally from other rapid, triggered fusion reactions. Emergency fusion might use a specialized protein and organelle emergency response team that can simultaneously promote impromptu homotypic fusion events between organelles and exocytic fusion events along the vertices between these fusion products and the plasma membrane.

  14. 13C-13C and 15N-13C correlation spectroscopy of membrane-associated and uniformly labeled human immunodeficiency virus and influenza fusion peptides: Amino acid-type assignments and evidence for multiple conformations

    NASA Astrophysics Data System (ADS)

    Bodner, Michele L.; Gabrys, Charles M.; Struppe, Jochem O.; Weliky, David P.

    2008-02-01

    Many viruses which cause disease including human immunodeficiency virus (HIV) and influenza are "enveloped" by a membrane and infection of a host cell begins with joining or "fusion" of the viral and target cell membranes. Fusion is catalyzed by viral proteins in the viral membrane. For HIV and for the influenza virus, these fusion proteins contain an ˜20-residue apolar "fusion peptide" that binds to target cell membranes and plays a critical role in fusion. For this study, the HIV fusion peptide (HFP) and influenza virus fusion peptide (IFP) were chemically synthesized with uniform C13, N15 labeling over large contiguous regions of amino acids. Two-dimensional C13-C13 and N15-C13 spectra were obtained for the membrane-bound fusion peptides and an amino acid-type C13 assignment was obtained for the labeled residues in HFP and IFP. The membrane used for the HFP sample had a lipid headgroup and cholesterol composition comparable to that of host cells of the virus, and the C13 chemical shifts were more consistent with β strand conformation than with helical conformation. The membrane used for the IFP sample did not contain cholesterol, and the chemical shifts of the dominant peaks were more consistent with helical conformation than with β strand conformation. There were additional peaks in the IFP spectrum whose shifts were not consistent with helical conformation. An unambiguous C13 and N15 assignment was obtained in an HFP sample with more selective labeling, and two shifts were identified for the Leu-9 CO, Gly-10 N, and Gly-10 Cα nuclei. These sets of two shifts may indicate two β strand registries such as parallel and antiparallel. Although most spectra were obtained on a 9.4T instrument, one C13-C13 correlation spectrum was obtained on a 16.4T instrument and was better resolved than the comparable 9.4T spectrum. More selective labeling and higher field may, therefore, be approaches to obtaining unambiguous assignments for membrane-associated fusion peptides.

  15. Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB.

    PubMed

    Tikhonova, Elena B; Devroy, Vishakha K; Lau, Sze Yi; Zgurskaya, Helen I

    2007-02-01

    Periplasmic membrane fusion proteins (MFPs) are essential components of the type I protein secretion systems and drug efflux pumps in Gram-negative bacteria. Previous studies suggested that MFPs connect the inner and outer membrane components of the transport systems and by this means co-ordinate the transfer of substrates across the two membranes. In this study, we purified and reconstituted the macrolide transporter MacAB from Escherichia coli. Here, MacA is a periplasmic MFP and MacB is an ABC-type transporter. Similar to other MFP-dependent transporters from E. coli, the in vivo function of MacAB requires the outer membrane channel TolC. The purified MacB displayed a basal ATPase activity in detergent micelles. This activity conformed to Michaelis-Menten kinetics but was unresponsive to substrates or accessory proteins. Upon reconstitution into proteoliposomes, the ATPase activity of MacB was strictly dependent on MacA. The catalytic efficiency of MacAB ATPase was more than 45-fold higher than the activity of MacB alone. Both the N- and C-terminal regions of MacA were essential for this activity. MacA stimulated MacB ATPase only in phospholipid bilayers and did not need the presence of macrolides. Our results suggest that MacA is a functional subunit of the MacB transporter. PMID:17214741

  16. Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

    PubMed

    Bedbrook, Claire N; Kato, Mihoko; Ravindra Kumar, Sripriya; Lakshmanan, Anupama; Nath, Ravi D; Sun, Fei; Sternberg, Paul W; Arnold, Frances H; Gradinaru, Viviana

    2015-08-20

    Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering. PMID:26211362

  17. Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

    PubMed

    Bedbrook, Claire N; Kato, Mihoko; Ravindra Kumar, Sripriya; Lakshmanan, Anupama; Nath, Ravi D; Sun, Fei; Sternberg, Paul W; Arnold, Frances H; Gradinaru, Viviana

    2015-08-20

    Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.

  18. Role of NF-kappa B in cell survival and transcription of latent membrane protein 1-expressing or Epstein-Barr virus latency III-infected cells.

    PubMed

    Cahir-McFarland, Ellen D; Carter, Kara; Rosenwald, Andreas; Giltnane, Jena M; Henrickson, Sarah E; Staudt, Louis M; Kieff, Elliott

    2004-04-01

    Epstein-Barr virus (EBV) latency III infection converts B lymphocytes into lymphoblastoid cell lines (LCLs) by expressing EBV nuclear and membrane proteins, EBNAs, and latent membrane proteins (LMPs), which regulate transcription through Notch and tumor necrosis factor receptor pathways. The role of NF-kappa B in LMP1 and overall EBV latency III transcriptional effects was investigated by treating LCLs with BAY11-7082 (BAY11). BAY11 rapidly and irreversibly inhibited NF-kappa B, decreased mitochondrial membrane potential, induced apoptosis, and altered LCL gene expression. BAY11 effects were similar to those of an NF-kappa B inhibitor, Delta N-I kappa B alpha, in effecting decreased JNK1 expression and in microarray analyses. More than 80% of array elements that decreased with Delta N-I kappa B alpha expression decreased with BAY11 treatment. Newly identified NF-kappa B-induced, LMP1-induced, and EBV-induced genes included pleckstrin, Jun-B, c-FLIP, CIP4, and I kappa B epsilon. Of 776 significantly changed array elements, 134 were fourfold upregulated in EBV latency III, and 74 were fourfold upregulated with LMP1 expression alone, whereas only 28 were more than fourfold downregulated by EBV latency III. EBV latency III-regulated gene products mediate cell migration (EBI2, CCR7, RGS1, RANTES, MIP1 alpha, MIP1 beta, CXCR5, and RGS13), antigen presentation (major histocompatibility complex proteins and JAW1), mitogen-activated protein kinase pathway (DUSP5 and p62Dok), and interferon (IFN) signaling (IFN-gamma R alpha, IRF-4, and STAT1). Comparison of EBV latency III LCL gene expression to immunoglobulin M (IgM)-stimulated B cells, germinal-center B cells, and germinal-center-derived lymphomas clustered LCLs with IgM-stimulated B cells separately from germinal-center cells or germinal-center lymphoma cells. Expression of IRF-2, AIM1, ASK1, SNF2L2, and components of IFN signaling pathways further distinguished EBV latency III-infected B cells from IgM-stimulated or

  19. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

    SciTech Connect

    Paquette, Stéphane G.; Banner, David; Chi, Le Thi Bao; Leon, Alberto J.; Xu, Luoling; Ran, Longsi; Huang, Stephen S.H.; Farooqui, Amber; and others

    2014-01-05

    Pandemic H1N1 influenza A (H1N1pdm) elicits stronger pulmonary inflammation than previously circulating seasonal H1N1 influenza A (sH1N1), yet mechanisms of inflammatory activation in respiratory epithelial cells during H1N1pdm infection are unclear. We investigated host responses to H1N1pdm/sH1N1 infection and virus entry mechanisms in primary human bronchial epithelial cells in vitro. H1N1pdm infection rapidly initiated a robust inflammatory gene signature (3 h post-infection) not elicited by sH1N1 infection. Protein secretion inhibition had no effect on gene induction. Infection with membrane fusion deficient H1N1pdm failed to induce robust inflammatory gene expression which was rescued with restoration of fusion ability, suggesting H1N1pdm directly triggered the inflammatory signature downstream of membrane fusion. Investigation of intra-virion components revealed H1N1pdm viral RNA (vRNA) triggered a stronger inflammatory phenotype than sH1N1 vRNA. Thus, our study is first to report H1N1pdm induces greater inflammatory gene expression than sH1N1 in vitro due to direct virus–epithelial cell interaction. - Highlights: • We investigated H1N1pdm/sH1N1 infection in primary epithelial cells. • H1N1pdm directly initiated a robust inflammatory gene signature, sH1N1 did not. • H1N1pdm viral RNA triggered a stronger response than sH1N1. • H1N1pdm induces greater response due to direct virus–cell interaction. • These results have potential to impact vaccine and therapeutic development.

  20. The fusion loops and membrane proximal region of Epstein-Barr virus glycoprotein B (gB) can function in the context of herpes simplex virus 1 gB when substituted individually but not in combination.

    PubMed

    Zago, Anna; Connolly, Sarah A; Spear, Patricia G; Longnecker, Richard

    2013-01-01

    Among the herpesvirus glycoprotein B (gB) fusion proteins, the hydrophobic content of fusion loops and membrane proximal regions (MPRs) are inversely correlated with each other. We examined the functional importance of the hydrophobicity of these regions by replacing them in herpes simplex virus type 1 gB with corresponding regions from Epstein-Barr virus gB. We show that fusion activity is dependent on the structural context in which the specific loops and MPR sequences exist, rather than a simple hydrophobic relationship.

  1. [Porous tarflen as a possible membrane material for membrane blood oxygenators. III. O2 and CO2 transport in the system modeling an artificial lung].

    PubMed

    Krajewska, B; Leszko, M

    1986-01-01

    Diffusional examinations of tarflen porous barriers of home make and porous teflon membranes of American make as well as selected nonporous membranes were performed in a system: O2--barrier--water + CO2, in order to evaluate the influence of aqueous phase on O2 and CO2 transport rate through barriers listed above. It was found that the effectiveness of O2 and CO2 exchange through the porous barriers in the examined system is controlled by O2 transport through the boundary water layer in contradistinction to the nonporous membranes. The effect of reduction of O2 and CO2 transport through the porous barriers, caused by the aqueous phase was noted. The higher the water pressure on a barrier the larger the effect is. Considerable water permeability of porous barriers as compared to that of nonporous membranes was stated. The results of the performed examinations indicate the usefulness of porous tarflen materials as a membrane material in membrane oxygenators of blood.

  2. Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

    PubMed

    Kim, Jin-Sik; Song, Saemee; Lee, Minho; Lee, Seunghwa; Lee, Kangseok; Ha, Nam-Chul

    2016-03-01

    The protein toxin HlyA of Escherichia coli is exported without a periplasmic intermediate by the type I secretion system (T1SS). The T1SS is composed of an inner membrane ABC transporter HlyB, an outer-membrane channel protein TolC, and a membrane fusion protein HlyD. However, the assembly of the T1SS remains to be elucidated. In this study, we determine the crystal structure of a part of the C-terminal periplasmic domain of HlyD. The long α-helical domain consisting of three α helices and a lipoyl domain was identified in the crystal structure. Based on the HlyD structure, we modeled the hexameric assembly of HlyD with a long α-helical barrel, which formed a complex with TolC in an intermeshing cogwheel-to-cogwheel manner, as observed in tripartite RND-type drug efflux pumps. These observations provide a structural blueprint for understanding the type I secretion system in pathogenic Gram-negative bacteria.

  3. The effects of La(III) on the peroxidation of membrane lipids in wheat seedling leaves under osmotic stress.

    PubMed

    Zeng, F; An, Y; Zhang, H; Zhang, M

    1999-08-01

    The physiological effects of the rare earth ion La3+ on the peroxidation of membrane lipids in wheat (Triticum aestivum L.) seedling leaves under osmotic stress were determined. With the passage of time under osmotic stress, the inhibition ability of lanthanum ions to the relative membrane permeability and concentration of malondialdehyde, superoxide radicals, and hydrogen peroxide caused by osmotic stress increased substantially, but no changes were noted in ferrous and relative water content. It indicated that lanthanum ions could not retain the water content because of osmotic stress. However, La3+ appears to decrease the production of *OH by reducing the content of O2*- and H2O2 of Haber-Weiss and Fenton reactions, which efficiently alleviated peroxidation of membrane lipids under osmotic stress and, to some degree, protected the membrane from injury of free radicals. Thus, La3+ increased the tolerance ability of plant to osmotic stress, which could assure the function of membrane normal temporally after stressed.

  4. Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa*

    PubMed Central

    Discola, Karen F.; Förster, Andreas; Boulay, François; Simorre, Jean-Pierre; Attree, Ina; Dessen, Andréa; Job, Viviana

    2014-01-01

    The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and β-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51–59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly. PMID:24297169

  5. Sequence homology and structural similarity between cytochrome b of mitochondrial complex III and the chloroplast b6-f complex: position of the cytochrome b hemes in the membrane.

    PubMed Central

    Widger, W R; Cramer, W A; Herrmann, R G; Trebst, A

    1984-01-01

    The amino acid sequences of cytochrome b of complex III from five different mitochondrial sources (human, bovine, mouse, yeast, and Aspergillus nidulans) and the chloroplast cytochrome b6 from spinach show a high degree of homology. Calculation of the distribution of hydrophobic residues with a "hydropathy" function that is conserved in this family of proteins implies that the membrane-folding pattern of the 42-kilodalton (kDa) mitochondrial cytochromes involves 8-9 membrane-spanning domains. The smaller 23-kDa chloroplast cytochrome appears to fold in five spanning domains that are similar to the first five of the mitochondria. Four highly conserved histidines are considered to be the likely ligands for the two hemes. The positions of the histidines along the spanning segments and in a cross section of the membrane-spanning alpha helices implies that two ligand pairs, His-82-His-197/198 and His-96-His-183, bridge the spanning peptides II and V, and the two hemes reside on opposite sides of the hydrophobic membrane core. In addition, the 17-kDa protein of the chloroplast b6-f complex appears to contain one or more of the functions of the COOH-terminal end of the mitochondrial cytochrome b polypeptide. PMID:6322162

  6. Hemagglutinin-neuraminidase enhances F protein-mediated membrane fusion of reconstituted Sendai virus envelopes with cells.

    PubMed Central

    Bagai, S; Puri, A; Blumenthal, R; Sarkar, D P

    1993-01-01

    Reconstituted Sendai virus envelopes containing both the fusion (F) protein and the hemagglutinin-neuraminidase (HN) (F,HN-virosomes) or only the F protein (F-virosomes) were prepared by solubilization of the intact virus with Triton X-100 followed by its removal by using SM2 Bio-Beads. Viral envelopes containing HN whose disulfide bonds were irreversibly reduced (HNred) were also prepared by treating the envelopes with dithiothreitol followed by dialysis (F,HNred-virosomes). Both F-virosomes and F,HNred-virosomes induced hemolysis of erythrocytes in the presence of wheat germ agglutinin, but the rates and extents were markedly lower than those for hemolysis induced by F,HN-virosomes. Using an assay based on the relief of self-quenching of a lipid probe incorporated in the Sendai virus envelopes, we demonstrate the fusion of both F,HN-virosomes and F-virosomes with cultured HepG2 cells containing the asialoglycoprotein receptor, which binds to a terminal galactose moiety of F. By desialylating the HepG2 cells, the entry mediated by HN-terminal sialic acid receptor interactions was bypassed. We show that both F-virosomes and F,HN-virosomes fuse with desialylated HepG2 cells, although the rate was two- to threefold higher if HN was included in the viral envelope. We also observed enhancement of fusion rates when both F and HN envelope proteins were attached to their specific receptors. Images PMID:8388501

  7. Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

    PubMed

    Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C

    2009-04-01

    Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

  8. Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes

    PubMed Central

    1975-01-01

    The presence in the Golgi fraction of glycoproteins destined to be incorporated into the microsomal membrane was investigated. When incubated in sucrose, washed Golgi vesicles released four major, weakly acidic glycoproteins, some of which could be incorporated into microsomal membranes by incubation. Double labeling with [3H]glucosamine and [14C]leucine demonstrated the incorporation of both protein and oligosaccharide moieties, and the main peak of radioactivity was associated with the 70,000 mol wt region after SDS- gel electrophoresis. The proteins that could be incorporated into microsomes were probably associated to a large extent with the outer surface of the Golgi membrane. Centrifugation of the proteins released from the Golgi in a KBr solution (p = 1.24) resulted in a separation of glycoproteins, those in the top layer most actively incorporated into microsomes. The lipoglycoproteins in the top layer that could be incorporated appeared in the 70,000 mol wt region after SDS-gel electrophoresis, as did the corresponding proteins isolated from the supernate. These results suggest that glycoproteins with completed oligosaccharide chains are released from the Golgi system to the cytosol and are subsequently transferred to microsomes as constitutive membrane components. PMID:1202020

  9. Solid-state nuclear magnetic resonance measurements of HIV fusion peptide 13CO to lipid 31P proximities support similar partially inserted membrane locations of the α helical and β sheet peptide structures.

    PubMed

    Gabrys, Charles M; Qiang, Wei; Sun, Yan; Xie, Li; Schmick, Scott D; Weliky, David P

    2013-10-01

    Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the ∼25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of "HFP", i.e., a ∼25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was (13)CO backbone labeled. Samples were then prepared that each contained a singly (13)CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric β sheet structure. Proximity between the HFP (13)CO nuclei and (31)P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct (13)CO shifts for the α helical and β sheet structures so that the proximities to (31)P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the (13)CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. "HFPmn" was a linear peptide that contained the 23 N-terminal residues of gp41. "HFPmn_V2E" contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and

  10. Solid-State Nuclear Magnetic Resonance Measurements of HIV Fusion Peptide 13CO to Lipid 31P Proximities Support Similar Partially Inserted Membrane Locations of the α Helical and β Sheet Peptide Structures

    NASA Astrophysics Data System (ADS)

    Gabrys, Charles M.; Qiang, Wei; Sun, Yan; Xie, Li; Schmick, Scott D.; Weliky, David P.

    2013-10-01

    Fusion of the human immunodeficiency virus (HIV) membrane and the host cell membrane is an initial step of infection of the host cell. Fusion is catalyzed by gp41, which is an integral membrane protein of HIV. The fusion peptide (FP) is the -25 N-terminal residues of gp41 and is a domain of gp41 that plays a key role in fusion catalysis likely through interaction with the host cell membrane. Much of our understanding of the FP domain has been accomplished with studies of -HFP-, i.e., a -25-residue peptide composed of the FP sequence but lacking the rest of gp41. HFP catalyzes fusion between membrane vesicles and serves as a model system to understand fusion catalysis. HFP binds to membranes and the membrane location of HFP is likely a significant determinant of fusion catalysis perhaps because the consequent membrane perturbation reduces the fusion activation energy. In the present study, many HFPs were synthesized and differed in the residue position that was 13CO backbone labeled. Samples were then prepared that each contained a singly 13CO labeled HFP incorporated into membranes that lacked cholesterol. HFP had distinct molecular populations with either α helical or oligomeric - sheet structure. Proximity between the HFP 13CO nuclei and 31P nuclei in the membrane headgroups was probed by solid-state NMR (SSNMR) rotational-echo double-resonance (REDOR) measurements. For many samples, there were distinct 13CO shifts for the α helical and - sheet structures so that the proximities to 31P nuclei could be determined for each structure. Data from several differently labeled HFPs were then incorporated into a membrane location model for the particular structure. In addition to the 13CO labeled residue position, the HFPs also differed in sequence and/or chemical structure. -HFPmn- was a linear peptide that contained the 23 N-terminal residues of gp41. -HFPmn_V2E- contained the V2E mutation that for HIV leads to greatly reduced extent of fusion and infection. The

  11. Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer

    PubMed Central

    Feng, Dongmei; Chen, Zhen; Wang, Zhiwen; Zhang, Chunlu; He, Kanglai; Guo, Shuyuan

    2015-01-01

    The insecticidal IE648 toxin is a truncated Cry1Ie protein with increased toxicity against Asian corn borer (ACB). Cry toxins are pore-forming toxins that disrupt insect midgut cells to kill the larvae. However, the peritrophic membrane (PM) is an important barrier that Cry toxins must cross before binding to midgut cells. Previously, it was shown that Cry toxins are able to bind and accumulate in the PM of several lepidopteran insects. Binding of IE648 toxin to PM of ACB was previously reported and the goal of the current work was the identification of the binding region between Cry1Ie and the PM of ACB. Homologous competition binding assays showed that this interaction was specific. Heterologous competition binding assays performed with different fragments corresponding to domain I, domain II and domain III allowed us to identify that domain III participates in the interaction of IE648 with the PM. Specifically, peptide D3-L8 (corresponding to Cry1Ie toxin residues 607 to 616), located in an exposed loop region of domain III is probably involved in this interaction. Ligand blot assays show that IE648 interact with chitin and PM proteins with sizes of 30, 32 and 80 kDa. The fact that domain III interacts with proteins of similar molecular masses supports that this region of the toxin might be involved in PM interaction. These data provide for the first time the identification of domain III as a putative binding region between PM and 3D-Cry toxin. PMID:26295704

  12. Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer.

    PubMed

    Feng, Dongmei; Chen, Zhen; Wang, Zhiwen; Zhang, Chunlu; He, Kanglai; Guo, Shuyuan

    2015-01-01

    The insecticidal IE648 toxin is a truncated Cry1Ie protein with increased toxicity against Asian corn borer (ACB). Cry toxins are pore-forming toxins that disrupt insect midgut cells to kill the larvae. However, the peritrophic membrane (PM) is an important barrier that Cry toxins must cross before binding to midgut cells. Previously, it was shown that Cry toxins are able to bind and accumulate in the PM of several lepidopteran insects. Binding of IE648 toxin to PM of ACB was previously reported and the goal of the current work was the identification of the binding region between Cry1Ie and the PM of ACB. Homologous competition binding assays showed that this interaction was specific. Heterologous competition binding assays performed with different fragments corresponding to domain I, domain II and domain III allowed us to identify that domain III participates in the interaction of IE648 with the PM. Specifically, peptide D3-L8 (corresponding to Cry1Ie toxin residues 607 to 616), located in an exposed loop region of domain III is probably involved in this interaction. Ligand blot assays show that IE648 interact with chitin and PM proteins with sizes of 30, 32 and 80 kDa. The fact that domain III interacts with proteins of similar molecular masses supports that this region of the toxin might be involved in PM interaction. These data provide for the first time the identification of domain III as a putative binding region between PM and 3D-Cry toxin. PMID:26295704

  13. Involvement of a membrane-bound class III adenylate cyclase in regulation of anaerobic respiration in Shewanella oneidensis MR-1.

    PubMed

    Charania, M A; Brockman, K L; Zhang, Y; Banerjee, A; Pinchuk, G E; Fredrickson, J K; Beliaev, A S; Saffarini, D A

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  14. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K. L.; Zhang, Y.; Banerjee, A.; Pinchuk, Grigoriy E.; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, Shewanella oneidensis MR-1 uses the cyclic AMP receptor protein (CRP) for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases, respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an Escherichia coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, dimethyl sulfoxide (DMSO), or Fe(III), whereas deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III) and, to a lesser extent, with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways, such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagellum biosynthesis, and electron transport were differentially expressed in the cyaC mutant but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration and may contribute to additional signaling pathways independent of CRP.

  15. Involvement of a Membrane-Bound Class III Adenylate Cyclase in Regulation of Anaerobic Respiration in Shewanella oneidensis MR-1

    SciTech Connect

    Charania, M.; Brockman, K.; Zhang, Yang; Banerjee, A.; Pinchuk, Grigoriy; Fredrickson, Jim K.; Beliaev, Alex S.; Saffarini, Daad

    2009-07-01

    Unlike other bacteria that use FNR to regulate anaerobic respiration, S. oneidensis MR-1 uses the cAMP receptor protein, CRP, for this purpose. Three putative genes, cyaA, cyaB, and cyaC, predicted to encode class I, class IV, and class III adenylate cyclases respectively, have been identified in the genome sequence of this bacterium. Functional validation through complementation of an E. coli cya mutant confirmed that these genes encode proteins with adenylate cyclase activities. Chromosomal deletion of either cyaA or cyaB did not affect anaerobic respiration with fumarate, DMSO, or Fe(III), whereas the deletion of cyaC caused deficiencies in respiration with DMSO and Fe(III), and to a lesser extent with fumarate. A phenotype similar to that of a crp mutant, which lacks the ability to grow anaerobically with DMSO, fumarate, and Fe(III), was obtained when both cyaA and cyaC were deleted. Microarray analysis of gene expression in the crp and the cyaC mutants revealed the involvement of both genes in the regulation of key respiratory pathways such as DMSO, fumarate, and Fe(III) reduction. Additionally, several genes associated with plasmid replication, flagella biosynthesis, and electron transport, were differentially expressed in the cyaC mutant, but not in the crp mutant. Our results indicated that CyaC plays a major role in regulating anaerobic respiration, and may contribute to additional signaling pathways independent of CRP.

  16. Two-Stage Mucogingival Surgery with Free Gingival Autograft and Biomend Membrane and Coronally Advanced Flap in Treatment of Class III Millers Recession.

    PubMed

    Rath, Avita; Varma, Smrithi; Paul, Renny

    2016-01-01

    Introduction. Gingival recession is an apical shift of the gingival margin with exposure of the root surface. This migration of the marginal tissue leads to esthetic concerns, dentin hypersensitivity, root caries, and cervical wear. It is, paradoxically, a common finding in patients with a high standard of oral hygiene, as well as in periodontally untreated populations with poor oral hygiene. Changing the topography of the marginal soft tissue in order to facilitate plaque control is a common indication for root coverage procedures and forms a major aspect of periodontal plastic surgeries. The regeneration of a new connective tissue attachment to denuded root surface is by allowing the selective coronal regrowth of periodontal ligament cells while excluding the gingival tissues from the root during wound healing by means of a barrier membrane. Case Presentation. This case reports a two-stage surgical technique for treatment of Miller's class III defect using free gingival autograft and type I absorbable collagen membrane (BioMend®, Zimmer Dental, USA)(§). Conclusions. The 6-month follow-up of the case showed a significant increase in attached gingiva suggesting it as a predictable alternative in the treatment of Millers class III defects. PMID:27525131

  17. Two-Stage Mucogingival Surgery with Free Gingival Autograft and Biomend Membrane and Coronally Advanced Flap in Treatment of Class III Millers Recession

    PubMed Central

    Paul, Renny

    2016-01-01

    Introduction. Gingival recession is an apical shift of the gingival margin with exposure of the root surface. This migration of the marginal tissue leads to esthetic concerns, dentin hypersensitivity, root caries, and cervical wear. It is, paradoxically, a common finding in patients with a high standard of oral hygiene, as well as in periodontally untreated populations with poor oral hygiene. Changing the topography of the marginal soft tissue in order to facilitate plaque control is a common indication for root coverage procedures and forms a major aspect of periodontal plastic surgeries. The regeneration of a new connective tissue attachment to denuded root surface is by allowing the selective coronal regrowth of periodontal ligament cells while excluding the gingival tissues from the root during wound healing by means of a barrier membrane. Case Presentation. This case reports a two-stage surgical technique for treatment of Miller's class III defect using free gingival autograft and type I absorbable collagen membrane (BioMend®, Zimmer Dental, USA)§. Conclusions. The 6-month follow-up of the case showed a significant increase in attached gingiva suggesting it as a predictable alternative in the treatment of Millers class III defects. PMID:27525131

  18. Spinal fusion

    MedlinePlus

    ... Anterior spinal fusion; Spine surgery - spinal fusion; Low back pain - fusion; Herniated disk - fusion ... If you had chronic back pain before surgery, you will likely still have some pain afterward. Spinal fusion is unlikely to take away all your pain ...

  19. Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant P. aeruginosa outer membrane protein F and outer membrane protein I fusion proteins.

    PubMed Central

    von Specht, B U; Knapp, B; Muth, G; Bröker, M; Hungerer, K D; Diehl, K D; Massarrat, K; Seemann, A; Domdey, H

    1995-01-01

    Recombinant outer membrane proteins (Oprs) of Pseudomonas aeruginosa were expressed in Escherichia coli as glutathione S-transferase (GST)-linked fusion proteins. GST-linked Oprs F and I (GST-OprF190-350 [GST linked to OprF spanning amino acids 190 to 350] and GST-OprI21-83, respectively) and recombinant hybrid Oprs (GST-OprF190-342-OprI21-83 and GST-OprI21-83-OprF190-350) were isolated and tested for their efficacy as vaccines in immunodeficient mice. GST-OprF-OprI protected the mice against a 975-fold 50% lethal dose of P. aeruginosa. Expression of GST-unfused OprF-OprI failed in E. coli, although this hybrid protein has been expressed without a fusion part in Saccharomyces cerevisiae and used for immunizing rabbits. The immune rabbit sera protected severe combined deficient (SCID) mice against a 1,000-fold 50% lethal dose of P. aeruginosa. Evidence is provided to show that the most C-terminal part of OprF (i.e., amino acids 332 to 350) carries an important protective epitope. Opr-based hybrid proteins may have implications for a clinical vaccine against P. aeruginosa. PMID:7729895

  20. Cellular Localization and Biochemical Characterization of a Chimeric Fluorescent Protein Fusion of Arabidopsis Cellulose Synthase-Like A2 Inserted into Golgi Membrane

    PubMed Central

    Lenucci, Marcello S.; Montefusco, Anna

    2014-01-01

    Cellulose synthase-like (Csl) genes are believed to encode enzymes for the synthesis of cell wall matrix polysaccharides. The subfamily of CslA is putatively involved in the biosynthesis of β-mannans. Here we report a study on the cellular localization and the enzyme activity of an Arabidopsis CslA family member, AtCslA2. We show that the fluorescent protein fusion AtCslA2-GFP, transiently expressed in tobacco leaf protoplasts, is synthesized in the ER and it accumulates in the Golgi stacks. The chimera is inserted in the Golgi membrane and is functional since membrane preparations obtained by transformed protoplasts carry out the in vitro synthesis of a 14C-mannan starting from GDP-d-[U-14C]mannose as substrate. The enzyme specific activity is increased by approximately 38% in the transformed protoplasts with respect to wild-type. Preliminary tests with proteinase K, biochemical data, and TM domain predictions suggest that the catalytic site of AtCslA2 faces the Golgi lumen. PMID:24558328

  1. The Gaussian Curvature Elastic Modulus of N-Monomethylated Dioleoylphosphatidylethanolamine: Relevance to Membrane Fusion and Lipid Phase Behavior

    PubMed Central

    Siegel, D. P.; Kozlov, M. M.

    2004-01-01

    The energy of intermediates in fusion of phospholipid bilayers is sensitive to \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\bar {{\\kappa}}}_{{\\mathrm{m}}},\\end{equation*}\\end{document} the saddle splay (Gaussian curvature) elastic modulus of the lipid monolayers. The value \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\bar {{\\kappa}}}_{{\\mathrm{m}}}\\end{equation*}\\end{document} is also important in understanding the stability of inverted cubic (QII) and rhombohedral (R) phases relative to the lamellar (Lα) and inverted hexagonal (HII) phases in phospholipids. However, \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\bar {{\\kappa}}}_{{\\mathrm{m}}}\\end{equation*}\\end{document} cannot be measured directly. It was previously measured by observing changes in QII phase lattice dimensions as a function of water content. Here we use observations of the phase behavior of N-mono-methylated dioleoylphosphatidylethanolamine (DOPE-Me) to determine \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\bar {{\\kappa}}}_{{\\mathrm{m}}}.\\end{equation*}\\end{document} At the temperature of the Lα/QII phase transition, TQ, the partial energies of the two phases are equal, and we can express \\documentclass[12pt

  2. Elimination of a pollution associated with chromic acid during the electro-deposition of Cr(III) using appropriate anodic and membrane materials in a double film bath.

    PubMed

    Jiang, Xiaojun; Chen, Wenchao; Xu, Hongbo

    2009-01-01

    A method using trivalent chromium has been used to replace hexavalent chromium for the electro-deposition of chromium. Using a tri-chamber bath system various anodic materials and membranes were evaluated to minimize the production of environmentally and health damaging chromic acid. By measuring the absorbance of Cr(VI) at 640 nm, the results indicate that the use of a titanium plated ruthenium (Ti-Ru) anode produces the least amount of chromic acid byproduct compared to lead-gold alloy and graphite anodes. The concentration of Cr(VI) in the immediate vicinity of the Ti-Ru anode decreased from 0.389 mg/L to 0 during a 40-min deposition period. The use of a Nafion(TM) quaternary cation exchange membrane portioning the buffer and anode selectively prevented Cr(III) from entering the anode compartment whilst allowing the migration of H(+) to maintain overall voltaic continuity. It has been demonstrated that the use of a Ti-Ru anode with a Nafion(TM) membrane can eliminate the production of chromic acid associated with the electro-deposition of chromium plate thereby preventing its health damaging exposure to plant operators and preventing discharge of Cr(VI) into the environment. Addition of a surfactant improved current efficiency by 34.7%.

  3. Snapshot of sequential SNARE assembling states between membranes shows that N-terminal transient assembly initializes fusion

    PubMed Central

    Wang, Yong Jian; Li, Feng; Rodriguez, Nicolas; Lafosse, Xavier; Gourier, Christine; Perez, Eric; Pincet, Frederic

    2016-01-01

    Many prominent biological processes are driven by protein assembling between membranes. Understanding the mechanisms then entails determining the assembling pathway of the involved proteins. Because the intermediates are by nature transient and located in the intermembrane space, this determination is generally a very difficult, not to say intractable, problem. Here, by designing a setup with sphere/plane geometry, we have been able to freeze one transient state in which the N-terminal domains of SNARE proteins are assembled. A single camera frame is sufficient to obtain the complete probability of this state with the transmembrane distance. We show that it forms when membranes are 20 nm apart and stabilizes by further assembling of the SNAREs at 8 nm. This setup that fixes the intermembrane distance, and thereby the transient states, while optically probing the level of molecular assembly by Förster resonance energy transfer (FRET) can be used to characterize any other transient transmembrane complexes. PMID:26979957

  4. Effects of alkyl alcohols and related chemicals on rat liver structure and function. III. Physiochemical properties of ethanol-, propanol- and butanol- treated rat liver mitochondrial membranes.

    PubMed

    Adachi, K; Momota, M; Teranishi, Y; Ueki, R; Hagiwara, M; Wakabayashi, T; Popinigis, J

    1992-08-01

    The physicochemical properties of mitochondria in liver tissue obtained from rats given 32% ethanol, 32% propanol or 6.9% butanol in drinking water for up to 3 months were investigated using differential scanning calorimetry and fluorescence polarization measurements. The results obtained were as follows: 1) Phospholipids extracted from mitochondria showed increases in the relative amounts of phosphatidylcholine, phosphatidylinositol and phosphatidylserine, and a decrease in the relative amount of phosphatidylethanolamine. An increase in the unsaturated/saturated fatty acid ratio of phospholipids was also observed. 2) Elevation of the thermotropic lipid phase transition temperature with a decrease in the enthalpy value (delta H) was revealed by differential scanning calorimetry. 3) The elevation of the lipid phase transition temperature was detected also by fluorescence polarization measurements using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Elevation of mitochondrial membrane fluidity was found in some of the experimental animals, but most showed no changes in comparison with the control. A possible role of membrane fusion in the mechanism of formation of ethanol-, propanol- and butanol-induced hepatic megamitochondria is discussed on the basis of these results.

  5. Investigation of the role of the BAM complex and SurA chaperone in outer-membrane protein biogenesis and type III secretion system expression in Salmonella.

    PubMed

    Fardini, Yann; Trotereau, Jérôme; Bottreau, Elisabeth; Souchard, Charlène; Velge, Philippe; Virlogeux-Payant, Isabelle

    2009-05-01

    In Escherichia coli, the assembly of outer-membrane proteins (OMP) requires the BAM complex and periplasmic chaperones, such as SurA or DegP. Previous work has suggested a potential link between OMP assembly and expression of the genes encoding type-III secretion systems. In order to test this hypothesis, we studied the role of the different lipoproteins of the BAM complex (i.e. BamB, BamC, BamD and BamE), and the periplasmic chaperones SurA and DegP, in these two phenotypes in Salmonella. Analysis of the corresponding deletion mutants showed that, as previously described with the DeltabamB mutant, BamD, SurA and, to a lesser extent, BamE play a role in outer-membrane biogenesis in Salmonella Enteritidis, while the membrane was not notably disturbed in DeltabamC and DeltadegP mutants. Interestingly, we found that BamD is not essential in Salmonella, unlike its homologues in Escherichia coli and Neisseria gonorrhoeae. In contrast, BamD was the only protein required for full expression of T3SS-1 and flagella, as demonstrated by transcriptional analysis of the genes involved in the biosynthesis of these T3SSs. In line with this finding, bamD mutants showed a reduced secretion of effector proteins by these T3SSs, and a reduced ability to invade HT-29 cells. As DeltasurA and DeltabamE mutants had lower levels of OMPs in their outer membrane, but showed no alteration in T3SS-1 and flagella expression, these results demonstrate the absence of a systematic link between an OMP assembly defect and the downregulation of T3SSs in Salmonella; therefore, this link appears to be related to a more specific mechanism that involves at least BamB and BamD. PMID:19372159

  6. Protoplast Fusion

    PubMed Central

    Yamada, Yasuyuki; Hara, Yasuhiro; Katagi, Hiroaki; Senda, Mitsugi

    1980-01-01

    The relation between the composition of the phospholipid molecular species in a cell membrane and the velocity of protoplast fusion was studied using cells cultured at a low temperature (10 C). Cells cultured at a low temperature contained larger proportions of phospholipids of low phase transition point, the 1,2-dilinoleoyl-type, than those cultured at a normal temperature (25 C). When treated with polyethylene glycol 6000, protoplasts from cells cultured at 10 C fused and progressed to the fused sphere stage more rapidly than did those from cells cultured at 25 C. PMID:16661339

  7. The Gaussian Curvature Elastic Modulus of N-Monomethylated Dioleoylphosphatidylethanolamine: Relevance to Membrane Fusion and Lipid Phase Behavior

    PubMed Central

    Siegel, D. P.; Kozlov, M. M.

    2004-01-01

    The energy of intermediates in fusion of phospholipid bilayers is sensitive to \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\bar {{\\kappa}}}_{{\\mathrm{m}}},\\end{equation*}\\end{document} the saddle splay (Gaussian curvature) elastic modulus of the lipid monolayers. The value \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\bar {{\\kappa}}}_{{\\mathrm{m}}}\\end{equation*}\\end{document} is also important in understanding the stability of inverted cubic (QII) and rhombohedral (R) phases relative to the lamellar (Lα) and inverted hexagonal (HII) phases in phospholipids. However, \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\bar {{\\kappa}}}_{{\\mathrm{m}}}\\end{equation*}\\end{document} cannot be measured directly. It was previously measured by observing changes in QII phase lattice dimensions as a function of water content. Here we use observations of the phase behavior of N-mono-methylated dioleoylphosphatidylethanolamine (DOPE-Me) to determine \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\bar {{\\kappa}}}_{{\\mathrm{m}}}.\\end{equation*}\\end{document} At the temperature of the Lα/QII phase transition, TQ, the partial energies of the two phases are equal, and we can express \\documentclass[12pt

  8. Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein.

    PubMed

    Mukai, Tetsu; Maeda, Yumi; Tamura, Toshiki; Matsuoka, Masanori; Tsukamoto, Yumiko; Makino, Masahiko

    2010-11-15

    To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells.

  9. [Enhanced activation of T lymphocytes by urease-deficient recombinant bacillus Calmette-Guérin producing heat shock protein 70-major membrane protein-II fusion protein].

    PubMed

    Makino, Masahiko; Mukai, Tetsu

    2012-09-01

    To activate naïve T cells convincingly using Mycobacterium bovis BCG (BCG), rBCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4+ and CD8+ subsets of naïve T cells than rBCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DC) to induce cytokine production and phenotypic changes, and activated CD4+ T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pre-treatment of DC with chloroquine inhibited both surface expression of MMP-II on DC and the activation of T cells by BCG-D70M-infected APCs. The naïve CD8+ T cell activation was inhibited by treatment of DC with brefeldin A and lactacystin so that the T cells was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naïve CD8+ T cells, effector T cells producing perforin and memory T cells having migration markers, were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70, and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II and urease depletion may provide useful tool for inducing better activation of naïve T cells.

  10. Avian sarcoma and leukosis virus-receptor interactions: From classical genetics to novel insights into virus-cell membrane fusion

    SciTech Connect

    Barnard, R.J.O.; Elleder, D.; Young, J.A.T. . E-mail: jyoung@salk.edu

    2006-01-05

    For over 40 years, avian sarcoma and leukosis virus (ASLV)-receptor interactions have been employed as a useful model system to study the mechanism of retroviral entry into cells. Pioneering studies on this system focused upon the genetic basis of the differential susceptibilities of different lines of chickens to infection by distinct subgroups of ASLV. These studies led to the definition of three distinct autosomal recessive genes that were predicted to encode cellular receptors for different viral subgroups. They also led to the concept of viral interference, i.e. the mechanism by which infection by one virus can render cells resistant to reinfection by other viruses that use the same cellular receptor. Here, we review the contributions that analyses of the ASLV-receptor system have made in unraveling the mechanisms of retroviral entry into cells and focus on key findings such as identification and characterization of the ASLV receptor genes and the subsequent elucidation of an unprecedented mechanism of virus-cell fusion. Since many of the initial findings on this system were published in the early volumes of Virology, this subject is especially well suited to this special anniversary issue of the journal.

  11. Efficient transport of Am(III) from nitric acid medium using a new conformationally constrained (N,N,N',N'-tetra-2-ethylhexyl)7-oxabicyclo[2.2.1]heptane-2,3-dicarboxamide across a supported liquid membrane.

    PubMed

    Sharma, S; Panja, S; Ghosh, S K; Dhami, P S; Gandhi, P M

    2016-03-15

    Am(III) is one of the most hazardous radionuclide present in nuclear fuel cycle. A new conformationally constrained diamide, (N,N,N',N'-tetra-2-ethylhexyl)7-oxabicyclo[2.2.1]heptane-2,3-dicarboxamide (OBDA) was studied for Am(III) transport from HNO3 medium across a Supported Liquid Membrane. Transport rate was observed to be significantly fast with ∼95% transport of Am(III) within 1h using 0.1M OBDA in the presence of 15% isodecyl alcohol (IDA)/n-dodecane as carrier. The mechanism of transport was investigated by studying various parameters like feed HNO3/NaNO3 concentration, OBDA concentration in the membrane, membrane pore size, membrane thickness etc. From these studies, the mechanism of transport was found to be diffusion controlled with diffusion co-efficient value of 5.1×10(-6)cm(2)/s. The membrane was found to be highly selective for tri- and tetra-valent actinides, and trivalent lanthanides. OBDA based membrane was found to be stable for at least for ten consecutive cycles of operation. PMID:26685064

  12. Virulence of Erwinia amylovora, a prevalent apple pathogen: Outer membrane proteins and type III secreted effectors increase fitness and compromise plant defenses.

    PubMed

    Holtappels, Michelle; Noben, Jean-Paul; Valcke, Roland

    2016-09-01

    Until now, no data are available on the outer membrane (OM) proteome of Erwinia amylovora, a Gram-negative plant pathogen, causing fire blight in most of the members of the Rosaceae family. Since the OM forms the interface between the bacterial cell and its environment it is in direct contact with the host. Additionally, the type III secretion system, embedded in the OM, is a pathogenicity factor of E. amylovora. To assess the influence of the OM composition and the secretion behavior on virulence, a 2D-DIGE analysis and gene expression profiling were performed on a high and lower virulent strain, both in vitro and in planta. Proteome data showed an increase in flagellin for the lower virulent strain in vitro, whereas, in planta several interesting proteins were identified as being differently expressed between both the strains. Further, gene expression of nearly all type III secreted effectors was elevated for the higher virulent strain, both in vitro and in planta. As a first, we report that several characteristics of virulence can be assigned to the OM proteome. Moreover, we demonstrate that secreted proteins prove to be the important factors determining differences in virulence between the strains, otherwise regarded as homogeneous on a genome level. PMID:27345300

  13. Improved laser-assisted vascular tissue fusion using solder-doped polymer membranes on a canine model

    NASA Astrophysics Data System (ADS)

    McNally-Heintzelman, Karen M.; Sorg, Brian S.; Hammer, Daniel X.; Heintzelman, Douglas L.; Hodges, Diane E.; Welch, Ashley J.

    2000-05-01

    Newly developed light-activated surgical adhesives have been investigated as a substitute to traditional protein solders for vascular tissue fusion without the need for sutures. Canine femoral arteries (n equals 14), femoral veins (n equals 14) and carotid arteries (n equals 10) were exposed, and a 0.3 to 0.6 cm longitudinal incision was made in the vessel walls. The surgical adhesive, composed of a poly(L-lactic-co-glycolic acid) scaffold doped with the traditional protein solder mix of bovine serum albumin and indocyanine green dye, was used to close the incisions in conjunction with an 805 nm diode laser. Blood flow was restored to the vessels immediately after the procedure and the incision sites were checked for patency. The new adhesives were flexible enough to be wrapped around the vessels while their solid nature avoided the problems associated with 'runaway' of the less viscous liquid protein solders widely used by researchers. Assessment parameters included measurement of the ex vivo intraluminal bursting pressure one to two hours after surgery, as well as histology. The acute intraluminal bursting pressures were significantly higher in the laser-solder group (greater than 300 mmHg) compared to the suture control group (less than 150 mmHg) where four evenly spaced sutures were used to repair the vessel (n equals 4). Histological analysis showed negligible evidence of collateral thermal damage to the underlying tissue in the laser-solder repair group. These initial results indicated that laser-assisted vascular repair using the new adhesives is safe, easy to perform, and contrary to conventional suturing, provides an immediate leak-free closure. In addition, the flexible and moldable nature of the new adhesives should allow them to be tailored to a wide range of tissue geometries, thus greatly improving the clinical applicability of laser-assisted tissue repair.

  14. Ultra-thin film composite mixed matrix membranes incorporating iron(III)-dopamine nanoparticles for CO2 separation.

    PubMed

    Kim, Jinguk; Fu, Qiang; Scofield, Joel M P; Kentish, Sandra E; Qiao, Greg G

    2016-04-21

    Iron dopamine nanoparticles (FeDA NPs) are incorporated into a nanoscale thick polyethylene glycol (PEG) matrix for the first time, to form ultra-thin film composite mixed matrix membranes (UTFC-MMMs) via a recently developed continuous assembly of polymers (CAP) nanotechnology. The FeDA NPs are prepared by in situ nano-complexation between Fe(3+) and DA and have a particle size that can be varied from 3 to 74 nanometers by adjusting the molar ratio of DA to Fe(3+) ion. The cross-linked selective layer with sub 100 nanometer thickness is prepared by atom transfer radical polymerisation of a mixture of PEG macrocross-linkers and FeDA NPs on top of a highly permeable poly(dimethyl siloxane) (PDMS) prelayer, which is spin-coated onto a porous polyacrylonitrile (PAN) substrate. The incorporation of the FeDA NPs within the PEG-based selective layer is confirmed by XPS analysis. The UTFC-MMMs (thickness: ∼45 nm) formed present excellent gas separation performance with a CO2 permeance of ∼1200 GPU (1 GPU = 10(-6) cm(3) (STP) cm(-2) s(-1) cmHg(-1)) and an enhanced CO2/N2 selectivity of over 35, which is the best performance for UTFC membranes in the reported literature.

  15. Serology of Neisseria gonorrhoeae: coagglutination serogroups WI and WII/III correspond to different outer membrane protein I molecules.

    PubMed Central

    Sandstrom, E G; Chen, K C; Buchanan, T M

    1982-01-01

    The 125I-labeled tryptic peptides of the outer membrane protein I of 33 previously characterized serological reference strains of Neisseria gonorrhoeae were investigated by peptide maps in relation to their coagglutination W serogroup. Serogroup WI strains tended to have lower-molecular-weight protein I molecules than did WII strains, and WIII strains had the highest-molecular-weight protein I molecules, although the serogroup could not be predicted from the molecular weights of the protein I molecules for a given strain. All 13 strains belonging to serogroup WI were found to have 11 peptides in common, as judged by their migration with respect to one another and to the internal marker valine in the peptide maps. Common peptides isolated from a given strain were found to comigrate with the corresponding common peptides from other strains in the same serogroup under various electrophoretic conditions. The 20 strains belonging to serogroups WII and WIII were all found to have 10 common peptides by the same criteria. When common peptides from serogroup WI were compared with the common peptides of serogroups WII and WIII, only three of these peptides appeared to be similar. Thus, two different outer membrane protein I molecules seem to exist which are mutually exclusive. Protein IA molecules contain the antigens recognized as serogroup WI, and protein IB molecules contain the antigens that characterize serogroups WII and WIII. Images PMID:6183215

  16. Ultra-thin film composite mixed matrix membranes incorporating iron(iii)-dopamine nanoparticles for CO2 separation

    NASA Astrophysics Data System (ADS)

    Kim, Jinguk; Fu, Qiang; Scofield, Joel M. P.; Kentish, Sandra E.; Qiao, Greg G.

    2016-04-01

    Iron dopamine nanoparticles (FeDA NPs) are incorporated into a nanoscale thick polyethylene glycol (PEG) matrix for the first time, to form ultra-thin film composite mixed matrix membranes (UTFC-MMMs) via a recently developed continuous assembly of polymers (CAP) nanotechnology. The FeDA NPs are prepared by in situ nano-complexation between Fe3+ and DA and have a particle size that can be varied from 3 to 74 nanometers by adjusting the molar ratio of DA to Fe3+ ion. The cross-linked selective layer with sub 100 nanometer thickness is prepared by atom transfer radical polymerisation of a mixture of PEG macrocross-linkers and FeDA NPs on top of a highly permeable poly(dimethyl siloxane) (PDMS) prelayer, which is spin-coated onto a porous polyacrylonitrile (PAN) substrate. The incorporation of the FeDA NPs within the PEG-based selective layer is confirmed by XPS analysis. The UTFC-MMMs (thickness: ~45 nm) formed present excellent gas separation performance with a CO2 permeance of ~1200 GPU (1 GPU = 10-6 cm3 (STP) cm-2 s-1 cmHg-1) and an enhanced CO2/N2 selectivity of over 35, which is the best performance for UTFC membranes in the reported literature.Iron dopamine nanoparticles (FeDA NPs) are incorporated into a nanoscale thick polyethylene glycol (PEG) matrix for the first time, to form ultra-thin film composite mixed matrix membranes (UTFC-MMMs) via a recently developed continuous assembly of polymers (CAP) nanotechnology. The FeDA NPs are prepared by in situ nano-complexation between Fe3+ and DA and have a particle size that can be varied from 3 to 74 nanometers by adjusting the molar ratio of DA to Fe3+ ion. The cross-linked selective layer with sub 100 nanometer thickness is prepared by atom transfer radical polymerisation of a mixture of PEG macrocross-linkers and FeDA NPs on top of a highly permeable poly(dimethyl siloxane) (PDMS) prelayer, which is spin-coated onto a porous polyacrylonitrile (PAN) substrate. The incorporation of the FeDA NPs within the PEG

  17. Binding of SEC11 indicates its role in SNARE recycling after vesicle fusion and identifies two pathways for vesicular traffic to the plasma membrane.

    PubMed

    Karnik, Rucha; Zhang, Ben; Waghmare, Sakharam; Aderhold, Christin; Grefen, Christopher; Blatt, Michael R

    2015-03-01

    SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.

  18. MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB-TolC, binds lipopolysaccharide core specifically and with high affinity.

    PubMed

    Lu, Shuo; Zgurskaya, Helen I

    2013-11-01

    The Escherichia coli MacAB-TolC transporter has been implicated in efflux of macrolide antibiotics and secretion of enterotoxin STII. In this study, we found that purified MacA, a periplasmic membrane fusion protein, contains one tightly bound rough core lipopolysaccharide (R-LPS) molecule per MacA molecule. R-LPS was bound specifically to MacA protein with affinity exceeding that of polymyxin B. Sequence analyses showed that MacA contains two high-density clusters of positively charged amino acid residues located in the cytoplasmic N-terminal domain and the periplasmic C-terminal domain. Substitutions in the C-terminal cluster reducing the positive-charge density completely abolished binding of R-LPS. At the same time, these substitutions significantly reduced the functionality of MacA in the protection of E. coli against macrolides in vivo and in the in vitro MacB ATPase stimulation assays. Taken together, our results suggest that R-LPS or a similar glycolipid is a physiological substrate of MacAB-TolC. PMID:23974027

  19. Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion

    NASA Astrophysics Data System (ADS)

    Yang, Sung-Tae; Kiessling, Volker; Tamm, Lukas K.

    2016-04-01

    Lipids and proteins are organized in cellular membranes in clusters, often called `lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of kBT to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors.

  20. Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion.

    PubMed

    Yang, Sung-Tae; Kiessling, Volker; Tamm, Lukas K

    2016-01-01

    Lipids and proteins are organized in cellular membranes in clusters, often called 'lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of kBT to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors. PMID:27113279

  1. NSOM/QD-based fluorescence-topographic image fusion directly reveals nano-spatial peak-valley polarities of CD69 and CD71 activation molecules on cell-membrane fluctuations during T-cell activation.

    PubMed

    Zhong, Liyun; Zhang, Zhun; Lu, Xiaoxu; Huang, Dan; Chen, Crystal Y; Wang, Richard; Chen, Zheng W

    2011-10-30

    Nano-spatial distribution of cell surface molecules on cell membrane fluctuations during T-cell activation has not been reported. In this study, we innovated application of near-field scanning optical microscopy (NSOM)/quantum dots (QDs)-based nanotechnology through three-dimensional image fusion algorithm to merge the simultaneously obtained dual-color fluorescence information and three-dimensional topography. This novel imaging system made it possible to visualize nano-spatial distribution and organization of early-activation molecules CD69 and late-activation molecules CD71 on cell-membrane fluctuations during T-cell activation. Interestingly, most CD69 molecules were clustered to form 250-500nm nano-domains polarizing predominantly in the peak of the cell-membrane fluctuations. In contrast, although CD71 molecules were also clustered as 250-500nm nano-domains, they polarized dominantly in the valley of the cell-membrane fluctuations. The peak-valley polarities of CD69 nano-domains and CD71 nano-domains implied their different functions. CD69 nano-domains polarizing on membrane-peak fluctuations might serve as transient platforms driving TCR/CD3-induced signaling and activation, whereas CD71 nano-domains distributing in the membrane-valley fluctuations appeared to facilitate iron uptake for increased metabolisms in T-cell activation. Importantly, this NSOM/QD-based fluorescence-topographic image fusion provides a powerful tool to visualize nano-spatial distribution of cell-surface molecules on cell-membrane fluctuations and enable better understanding of distribution-function relationship.

  2. Lipids implicated in the journey of a secretory granule: from biogenesis to fusion.

    PubMed

    Tanguy, Emeline; Carmon, Ophélie; Wang, Qili; Jeandel, Lydie; Chasserot-Golaz, Sylvette; Montero-Hadjadje, Maité; Vitale, Nicolas

    2016-06-01

    The regulated secretory pathway begins with the formation of secretory granules by budding from the Golgi apparatus and ends by their fusion with the plasma membrane leading to the release of their content into the extracellular space, generally following a rise in cytosolic calcium. Generation of these membrane-bound transport carriers can be classified into three steps: (i) cargo sorting that segregates the cargo from resident proteins of the Golgi apparatus, (ii) membrane budding that encloses the cargo and depends on the creation of appropriate membrane curvature, and (iii) membrane fission events allowing the nascent carrier to separate from the donor membrane. These secretory vesicles then mature as they are actively transported along microtubules toward the cortical actin network at the cell periphery. The final stage known as regulated exocytosis involves the docking and the priming of the mature granules, necessary for merging of vesicular and plasma membranes, and the subsequent partial or total release of the secretory vesicle content. Here, we review the latest evidence detailing the functional roles played by lipids during secretory granule biogenesis, recruitment, and exocytosis steps. In this review, we highlight evidence supporting the notion that lipids play important functions in secretory vesicle biogenesis, maturation, recruitment, and membrane fusion steps. These effects include regulating various protein distribution and activity, but also directly modulating membrane topology. The challenges ahead to understand the pleiotropic functions of lipids in a secretory granule's journey are also discussed. This article is part of a mini review series on Chromaffin cells (ISCCB Meeting, 2015).

  3. Water in barnacle muscle. III. NMR studies of fresh fibers and membrane-damaged fibers equilibrated with selected solutes.

    PubMed Central

    Burnell, E E; Clark, M E; Hinke, J A; Chapman, N R

    1981-01-01

    Water in barnacle muscle has been studied using NMR techniques. Fresh fibers are compared with membrane-damaged fibers treated with solutes that greatly alter fixed charge and total water content. Both water (97%) and solute (3%) protons are visible in continuous wave spectra of oriented fresh fibers. No local field inhomogeneities were detected, nor are cell solutes significantly bound. In pulse experiments, all cell water is visible and exhibits a single exponential decay. In fresh fibers, T2 approximately or equal to 40 ms; faster decaying signals are assigned to immobile and mobile protons on macromolecules. T1 and T1p are frequency dependent. Using equations derived for a two-compartment model with fast exchange, we calculate the following: tau b, the correlation time for anisotropic rotational motion of bound water; Sb, its order parameter; tau ex, the correlation time for exchange between bound and free fractions; f, the fraction of water bound; and Hr, the grams of water bound per gram of macromolecule. Whereas f varies inversely with total water content, the other parameters are virtually constant, with values: tau b approximately or equal to 1.3 X 10(-8) S; tau ex approximately or equal to 8 X 10(-6) s; Sb approximately or equal to 0.06; and Hr approximately or equal to 0.1g H2O/g macromolecule. Thus, the NMR relaxation detectable properties of water bound to macromolecules are unaffected by solutes that greatly alter the macromolecular surface charge. PMID:7272435

  4. Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein

    PubMed Central

    Vassell, Russell; He, Yong; Vennakalanti, Prasad; Dey, Antu K.; Zhuang, Min; Wang, Wei; Sun, Yide; Biron-Sorek, Zohar; Srivastava, Indresh K.; LaBranche, Celia C.; Montefiori, David C.; Barnett, Susan W.; Weiss, Carol D.

    2015-01-01

    The membrane proximal external region (MPER) of the gp41 subunit of the HIV-1 envelope glycoprotein (Env) contains determinants for broadly neutralizing antibodies and has remained an important focus of vaccine design. However, creating an immunogen that elicits broadly neutralizing antibodies to this region has proven difficult in part due to the relative inaccessibility of the MPER in the native conformation of Env. Here, we describe the antigenicity and immunogenicity of a panel of oligomeric gp41 immunogens designed to model a fusion-intermediate conformation of Env in order to enhance MPER exposure in a relevant conformation. The immunogens contain segments of the gp41 N- and C-heptad repeats to mimic a trapped intermediate, followed by the MPER, with variations that include different N-heptad lengths, insertion of extra epitopes, and varying C-termini. These well-characterized immunogens were evaluated in two different immunization protocols involving gp41 and gp140 proteins, gp41 and gp160 DNA primes, and different immunization schedules and adjuvants. We found that the immunogens designed to reduce extension of helical structure into the MPER elicited the highest MPER antibody binding titers, but these antibodies lacked neutralizing activity. The gp41 protein immunogens also elicited higher MPER titers than the gp140 protein immunogen. In prime-boost studies, the best MPER responses were seen in the groups that received DNA priming with gp41 vectors followed by gp41 protein boosts. Finally, although titers to the entire protein immunogen were similar in the two immunization protocols, MPER-specific titers differed, suggesting that the immunization route, schedule, dose, or adjuvant may differentially influence MPER immunogenicity. These findings inform the design of future MPER immunogens and immunization protocols. PMID:26087072

  5. Big fusion, little fusion

    NASA Astrophysics Data System (ADS)

    Chen, Frank; ddtuttle

    2016-08-01

    In reply to correspondence from George Scott and Adam Costley about the Physics World focus issue on nuclear energy, and to news of construction delays at ITER, the fusion reactor being built in France.

  6. The modular structure of the inner-membrane ring component PrgK facilitates assembly of the type III secretion system basal body.

    PubMed

    Bergeron, Julien R C; Worrall, Liam J; De, Soumya; Sgourakis, Nikolaos G; Cheung, Adrienne H; Lameignere, Emilie; Okon, Mark; Wasney, Gregory A; Baker, David; McIntosh, Lawrence P; Strynadka, Natalie C J

    2015-01-01

    The type III secretion system (T3SS) is a large macromolecular assembly found at the surface of many pathogenic Gram-negative bacteria. Its role is to inject toxic "effector" proteins into the cells of infected organisms. The molecular details of the assembly of this large, multimembrane-spanning complex remain poorly understood. Here, we report structural, biochemical, and functional analyses of PrgK, an inner-membrane component of the prototypical Salmonella typhimurium T3SS. We have obtained the atomic structures of the two ring building globular domains and show that the C-terminal transmembrane helix is not essential for assembly and secretion. We also demonstrate that structural rearrangement of the two PrgK globular domains, driven by an interconnecting linker region, may promote oligomerization into ring structures. Finally, we used electron microscopy-guided symmetry modeling to propose a structural model for the intimately associated PrgH-PrgK ring interaction within the assembled basal body. PMID:25533490

  7. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice

    SciTech Connect

    Valdes, Iris; Bernardo, Lidice; Pavon, Alekis; Guzman, Maria G.

    2009-11-25

    Based on the immunogenicity of domain III from the Envelope protein of dengue virus as well as the proven protective capacity of the capsid antigen, we have designed a novel domain III-capsid chimeric protein with the goal of obtaining a molecule potentially able to induce both humoral and cell-mediated immunity (CMI). After expression of the recombinant gene in Escherichia coli, the domain III moiety retained its antigenicity as evaluated with anti-dengue sera. In order to explore alternatives for modulating the immunogenicity of the protein, it was mixed with oligodeoxynucleotides in order to obtain particulated aggregates and then immunologically evaluated in mice in comparison with non-aggregated controls. Although the humoral immune response induced by both forms of the protein was equivalent, the aggregated variant resulted in a much stronger CMI as measured by in vitro IFN-gamma secretion and protection experiments, mediated by CD4{sup +} and CD8{sup +} cells. The present work provides additional evidence in support for a crucial role of CMI in protection against dengue virus and describes a novel vaccine candidate against the disease based on a recombinant protein that can stimulate both arms of the acquired immune system.

  8. Pharmacophore determination of a gp120 C terminal-derived anti-HIV peptide construct interfering with membrane fusion suggesting that processing of the gp120 C terminus is a prelude to fusion.

    PubMed

    Barbouche, R; Feyfant, E; Belhaj, B; Fenouillet, E

    2002-02-10

    A multiple antigen peptide [CLIV; (PTKAKRR1VVQREKR2)4-K2-K-betaA] from the C terminus of the gp120 subunit of HIV Env inhibits Env-mediated cell-to-cell fusion through direct interference with the process (Virology 2000;273:169). We have examined various CLIV analogs using a cell-to-cell fusion assay, receptor binding assays, and molecular modeling to further address the characteristics of the peptide responsible for its anti-HIV activity. We show that (1) CLIV does not interfere with Env binding to CD4 and does not interact with the binding site of Env on CXCR4; (2) CLIV does not inhibit protease activities already reported to play a role in fusion; and (3) the pharmacophore is composed of cleavage site1 with amino acid residues at its C terminal end. Based on our data and on the literature, we propose that CLIV interferes with processing of the gp120 C terminus at site1 by the lymphocyte surface after CD4 binding. Our hypothesis implies that the cleavage region of Env is submitted to a stepwise processing including the known intracellular cleavage of gp160 at site2 in order to set the activation of the fusion peptide and a yet unexplored cleavage at site1 by the target cell surface that triggers fusion.

  9. Role for the disulfide-bonded region of human immunodeficiency virus type 1 gp41 in receptor-triggered activation of membrane fusion function

    SciTech Connect

    Bellamy-McIntyre, Anna K.; Baer, Severine; Ludlow, Louise; Drummer, Heidi E.; Poumbourios, Pantelis

    2010-04-16

    The conserved disulfide-bonded region (DSR) of the human immunodeficiency virus type 1 (HIV-1) fusion glycoprotein, gp41, mediates association with the receptor-binding glycoprotein, gp120. Interactions between gp120, CD4 and chemokine receptors activate the fusion activity of gp41. The introduction of W596L and W610F mutations to the DSR of HIV-1{sub QH1549.13} blocked viral entry and hemifusion without affecting gp120-gp41 association. The fusion defect correlated with inhibition of CD4-triggered gp41 pre-hairpin formation, consistent with the DSR mutations having decoupled receptor-induced conformational changes in gp120 from gp41 activation. Our data implicate the DSR in sensing conformational changes in the gp120-gp41 complex that lead to fusion activation.

  10. Fusion of Enveloped Viruses in Endosomes.

    PubMed

    White, Judith M; Whittaker, Gary R

    2016-06-01

    Ari Helenius launched the field of enveloped virus fusion in endosomes with a seminal paper in the Journal of Cell Biology in 1980. In the intervening years, a great deal has been learned about the structures and mechanisms of viral membrane fusion proteins as well as about the endosomes in which different enveloped viruses fuse and the endosomal cues that trigger fusion. We now recognize three classes of viral membrane fusion proteins based on structural criteria and four mechanisms of fusion triggering. After reviewing general features of viral membrane fusion proteins and viral fusion in endosomes, we delve into three characterized mechanisms for viral fusion triggering in endosomes: by low pH, by receptor binding plus low pH and by receptor binding plus the action of a protease. We end with a discussion of viruses that may employ novel endosomal fusion-triggering mechanisms. A key take-home message is that enveloped viruses that enter cells by fusing in endosomes traverse the endocytic pathway until they reach an endosome that has all of the environmental conditions (pH, proteases, ions, intracellular receptors and lipid composition) to (if needed) prime and (in all cases) trigger the fusion protein and to support membrane fusion.

  11. Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

    PubMed

    Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

    2011-11-22

    Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41.

  12. Fe(III) and Fe(II) ions different effects on Enterococcus hirae cell growth and membrane-associated ATPase activity

    SciTech Connect

    Vardanyan, Zaruhi; Trchounian, Armen

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Fe{sup 3+} stimulates but Fe{sup 2+} suppresses Enterococcus hirae wild-type and atpD mutant growth. Black-Right-Pointing-Pointer Fe ions change oxidation-reduction potential drop during cell growth. Black-Right-Pointing-Pointer Fe{sup 3+} and Fe{sup 2+} have opposite effects on a membrane-associated ATPase activity. Black-Right-Pointing-Pointer These effects are either in the presence of F{sub 0}F{sub 1} inhibitor or non-functional F{sub 0}F{sub 1}. Black-Right-Pointing-Pointer Fe ions decrease protons and coupled potassium ions fluxes across the membrane. -- Abstract: Enterococcus hirae is able to grow under anaerobic conditions during glucose fermentation (pH 8.0) which is accompanied by acidification of the medium and drop in its oxidation-reduction potential (E{sub h}) from positive values to negative ones (down to {approx}-200 mV). In this study, iron (III) ions (Fe{sup 3+}) have been shown to affect bacterial growth in a concentration-dependent manner (within the range of 0.05-2 mM) by decreasing lag phase duration and increasing specific growth rate. While iron(II) ions (Fe{sup 2+}) had opposite effects which were reflected by suppressing bacterial growth. These ions also affected the changes in E{sub h} values during bacterial growth. It was revealed that ATPase activity with and without N,N Prime -dicyclohexylcarbodiimide (DCCD), an inhibitor of the F{sub 0}F{sub 1}-ATPase, increased in the presence of even low Fe{sup 3+} concentration (0.05 mM) but decreased in the presence of Fe{sup 2+}. It was established that Fe{sup 3+} and Fe{sup 2+} both significantly inhibited the proton-potassium exchange of bacteria, but stronger effects were in the case of Fe{sup 2+} with DCCD. Such results were observed with both wild-type ATCC9790 and atpD mutant (with defective F{sub 0}F{sub 1}) MS116 strains but they were different with Fe{sup 3+} and Fe{sup 2+}. It is suggested that the effects of Fe{sup 3+} might be due to

  13. Kinetics of Reduction of Fe(III) Complexes by Outer Membrane Cytochromes MtrC and OmcA of Shewanella oneidensis MR-1

    SciTech Connect

    Wang, Zheming; Liu, Chongxuan; Wang, Xuelin; Marshall, Matthew J.; Zachara, John M.; Rosso, Kevin M.; Dupuis, Michel; Fredrickson, Jim K.; Heald, Steve M.; Shi, Liang

    2008-09-12

    Shewanella Oneidensis MR-1 possesses up to 42 c-type cytochromes with heme content varying between 1 to as many as 37. Among them, the outer-membrane cytochromes, particularly MtrC and OmcA, are suspected to function as terminal reductases and are responsible for its enzymatic catalysis capability. So far, the mechanisms of metal reduction by these outer-membrane cytochromes are unknown. In this work, we report the study of reduction kinetics of a series of Fe(III) complexes with citrate, NTA and EDTA by abiotically reduced MtrC and OmcA using a stopped-flow technique in combination with theoretical computation methods within the framework of the electron transfer theory of Marcus and speciation calculations based on the current thermodynamic database. Stopped-flow kinetic data showed that the reaction was very fast and appeared to proceed in two stages, a fast stage that completes in much less than a second and a slower stage afterwards. For a given complex, the reaction is faster by reduction with MtrC than OmcA, while for a given protein, the reaction completes in the decreasing order of Fe-EDTA > Fe-NTA > Fe-citrate. All the stopped-flow kinetic curves could be modeled by two parallel second-order bimolecular redox reactions with second-order rate constants ranging from 0.872 µM-1s-1 for the fast reaction between MtrC with Fe-EDTA complex to 0.012 µM-1s-1 for the slow reaction between OmcA and Fe-citrate complex. Speciation calculations indicated that at both metal:ligand ratios, 1:1.5 and 1:10, a single dominant ferric complex was responsible for the observed reaction for each ligand and, therefore, the observed dual-reaction pathways was attributed to the differences in the reduction behavior among various heme groups within each protein. The results of redox potential calculations with known thermodynamic data show only small differences on the scale of a few millivolts among the three complexes, suggested that

  14. Nuclear Fusion

    NASA Astrophysics Data System (ADS)

    Veres, G.

    This chapter is devoted to the fundamental concepts of nuclear fusion. To be more precise, it is devoted to the theoretical basics of fusion reactions between light nuclei such as hydrogen, helium, boron, and lithium. The discussion is limited because our purpose is to focus on laboratory-scale fusion experiments that aim at gaining energy from the fusion process. After discussing the methods of calculating the fusion cross section, it will be shown that sustained fusion reactions with energy gain must happen in a thermal medium because, in beam-target experiments, the energy of the beam is randomized faster than the fusion rate. Following a brief introduction to the elements of plasma physics, the chapter is concluded with the introduction of the most prominent fusion reactions ongoing in the Sun.

  15. Functional homology of gHs and gLs from EBV-related {gamma}-herpesviruses for EBV-induced membrane fusion

    SciTech Connect

    Omerovic, Jasmina; Longnecker, Richard . E-mail: r-longnecker@northwestern.edu

    2007-08-15

    Epstein-Barr virus (EBV) is a human {gamma}-herpesvirus that primarily infects B lymphocytes and epithelial cells. Entry of EBV into B cells requires the viral glycoproteins gp42, gH/gL and gB, while gp42 is not necessary for infection of epithelial cells. In EBV, gH and gL form two distinct complexes, a bipartite complex that contains only gH and gL, used for infection of epithelial cells, and a tripartite complex that additionally includes gp42, used for infection of B cells. The gH/gL complex is conserved within the herpesvirus family, but its exact role in entry and mechanism of fusion is not yet known. To understand more about the functionality of EBVgH/gL, we investigated the functional homology of gHs and gLs from human herpesvirus 8 (HHV8) and two primate (rhesus and marmoset) {gamma}-herpesviruses in EBV-mediated virus-free cell fusion assay. Overall, gHs and gLs from the more homologous primate herpesviruses were better at complementing EBV gH and gL in fusion than HHV8 gH and gL. Interestingly, marmoset gH was able to complement fusion with epithelial cells, but not B cells. Further investigation of this led to the discovery that EBVgH is the binding partner of gp42 in the tripartite complex and the absence of fusion with B cells in the presence of marmoset gH/gL is due to its inability to bind gp42.

  16. Specific interaction of CXCR4 with CD4 and CD8{alpha}: Functional analysis of the CD4/CXCR4 interaction in the context of HIV-1 envelope glycoprotein-mediated membrane fusion

    SciTech Connect

    Basmaciogullari, Stephane . E-mail: basmaciogullari@cochin.inserm.fr; Pacheco, Beatriz; Bour, Stephan; Sodroski, Joseph

    2006-09-15

    We investigated possible interactions between HIV-1 receptor (CD4) and the main coreceptors CXCR4 and CCR5. We found that CD4 and CXCR4 coexpressed in 293T cells form a complex that can be immunoprecipitated with antibodies directed against the extracellular domain of either protein. Mutagenesis revealed that the CD4/CXCR4 interaction maps to two previously uncharacterized basic motifs in the cytoplasmic domain of CD4. HIV-1 envelope glycoprotein-mediated membrane fusion was found to be independent of the ability of CD4 and CXCR4 to interact, whether fusion was studied in a virus-cell or a cell-cell model. However, this interaction might explain the adaptation of HIV-1 to CXCR4 as an alternative to CCR5. We found that CXCR4 also interacts with the cytoplasmic domain of CD8{alpha} in a way that is similar to the CD4/CXCR4 interaction. The CD4/CXCR4 and CD8{alpha}/CXCR4 interactions may thus be involved in cellular signaling pathways shared by the CD4 and CD8{alpha} molecules.

  17. Fusion Implementation

    SciTech Connect

    J.A. Schmidt

    2002-02-20

    If a fusion DEMO reactor can be brought into operation during the first half of this century, fusion power production can have a significant impact on carbon dioxide production during the latter half of the century. An assessment of fusion implementation scenarios shows that the resource demands and waste production associated with these scenarios are manageable factors. If fusion is implemented during the latter half of this century it will be one element of a portfolio of (hopefully) carbon dioxide limiting sources of electrical power. It is time to assess the regional implications of fusion power implementation. An important attribute of fusion power is the wide range of possible regions of the country, or countries in the world, where power plants can be located. Unlike most renewable energy options, fusion energy will function within a local distribution system and not require costly, and difficult, long distance transmission systems. For example, the East Coast of the United States is a prime candidate for fusion power deployment by virtue of its distance from renewable energy sources. As fossil fuels become less and less available as an energy option, the transmission of energy across bodies of water will become very expensive. On a global scale, fusion power will be particularly attractive for regions separated from sources of renewable energy by oceans.

  18. Modeling the effect of charge density in the active layers of reverse osmosis and nanofiltration membranes on the rejection of arsenic(III) and potassium iodide.

    PubMed

    Coronell, Orlando; Mi, Baoxia; Mariñas, Benito J; Cahill, David G

    2013-01-01

    We used an extended solution-diffusion model that incorporates Donnan electrostatic exclusion of ions and unhindered advection due to imperfections, and measurements of charge density in the polyamide active layers of reverse osmosis (RO) and nanofiltration (NF) membranes, to predict the rejection of a strong electrolyte (i.e., potassium iodide) and a weak acid (i.e., arsenious acid) as a function of the pH of the feed aqueous solution. Predictions of solute rejection were in agreement with experimental data indicating that (i) the extended solution-diffusion model taking into account Donnan exclusion and unhindered advection due to imperfections satisfactorily describes the effect of pH on solute rejection by RO/NF membranes and (ii) measurement of charge density in active layers provides a valuable characterization of RO/NF membranes. Our results and analysis also indicate that independent ions, and not ion pairs, dominate the permeation of salts. PMID:23199291

  19. Image fusion

    NASA Technical Reports Server (NTRS)

    Pavel, M.

    1993-01-01

    The topics covered include the following: a system overview of the basic components of a system designed to improve the ability of a pilot to fly through low-visibility conditions such as fog; the role of visual sciences; fusion issues; sensor characterization; sources of information; image processing; and image fusion.

  20. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    SciTech Connect

    Bernard, Eric; Simmons, Graham; Chazal, Nathalie; and others

    2015-02-15

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

  1. Pseudorevertants of a Semliki Forest Virus Fusion-Blocking Mutation Reveal a Critical Interchain Interaction in the Core Trimer▿

    PubMed Central

    Liu, Catherine Y.; Besanceney, Christen; Song, Yifan; Kielian, Margaret

    2010-01-01

    Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells by a low-pH-triggered membrane fusion reaction mediated by the viral E1 protein. E1 inserts into target membranes and refolds to a hairpin-like homotrimer containing a central core trimer and an outer layer composed of domain III and the juxtamembrane stem region. The key residues involved in mediating E1 trimerization are not well understood. We recently showed that aspartate 188 in the interface of the core trimer plays a critical role. Substitution with lysine (D188K) blocks formation of the core trimer and E1 trimerization and strongly inhibits virus fusion and infection. Here, we have isolated and characterized revertants that rescued the fusion and growth defects of D188K. These revertants included pseudorevertants containing acidic or polar neutral residues at E1 position 188 and a second-site revertant containing an E1 K176T mutation. Computational analysis using multiconformation continuum electrostatics revealed an important interaction bridging D188 of one chain with K176 of the adjacent chain in the core trimer. E1 K176 is completely conserved among the alphaviruses, and mutations of K176 to threonine (K176T) or isoleucine (K176I) produced similar fusion phenotypes as D188 mutants. Together, our data support a model in which a ring of three salt bridges formed by D188 and K176 stabilize the core trimer, a key intermediate of the alphavirus fusion protein. PMID:20826687

  2. Control of mechanically activated polymersome fusion: Factors affecting fusion

    SciTech Connect

    Henderson, Ian M.; Paxton, Walter F.

    2014-12-15

    Previously we have studied the mechanically-activated fusion of extruded (200 nm) polymer vesicles into giant polymersomes using agitation in the presence of salt. In this study we have investigated several factors contributing to this phenomenon, including the effects of (i) polymer vesicle concentration, (ii) agitation speed and duration, and iii) variation of the salt and its concentration. It was found that increasing the concentration of the polymer dramatically increases the production of giant vesicles through the increased collisions of polymersomes. Our investigations also found that increasing the frequency of agitation increased the efficiency of fusion, though ultimately limited the size of vesicle which could be produced due to the high shear involved. Finally it was determined that salt-mediation of the fusion process was not limited to NaCl, but is instead a general effect facilitated by the presence of solvated ionic compounds, albeit with different salts initiating fusion at different concentration.

  3. Control of mechanically activated polymersome fusion: Factors affecting fusion

    DOE PAGESBeta

    Henderson, Ian M.; Paxton, Walter F.

    2014-12-15

    Previously we have studied the mechanically-activated fusion of extruded (200 nm) polymer vesicles into giant polymersomes using agitation in the presence of salt. In this study we have investigated several factors contributing to this phenomenon, including the effects of (i) polymer vesicle concentration, (ii) agitation speed and duration, and iii) variation of the salt and its concentration. It was found that increasing the concentration of the polymer dramatically increases the production of giant vesicles through the increased collisions of polymersomes. Our investigations also found that increasing the frequency of agitation increased the efficiency of fusion, though ultimately limited the sizemore » of vesicle which could be produced due to the high shear involved. Finally it was determined that salt-mediation of the fusion process was not limited to NaCl, but is instead a general effect facilitated by the presence of solvated ionic compounds, albeit with different salts initiating fusion at different concentration.« less

  4. Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion

    PubMed Central

    Yang, Sung-Tae; Kiessling, Volker; Tamm, Lukas K.

    2016-01-01

    Lipids and proteins are organized in cellular membranes in clusters, often called ‘lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of kBT to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors. PMID:27113279

  5. In vitro fusion of Acanthamoeba phagolysosomes. I. Demonstration and quantitation of vacuole fusion in Acanthamoeba homogenates.

    PubMed

    Oates, P J; Touster, O

    1976-02-01

    Fusion of phagolysosomes (PLs) has been demonstrated to occur in vitro. Two separate cell homogenates of the ameba Acanthamoeba sp. (Neff) were prepared, each rich in PLs labeled with distinctive particulate markers. Portions of each were incubated together in vitro and fusion occurred as evidenced by the appearance of PLs containing both types of markers. Fusion was confirmed by electron microscopy, including serial sectioning. The membranes of fused vacuoles excluded the dye eosin Y. Surviving cells in the homogenates were not responsible for the observed fusion. Fusion was obtained using either synthetic markers (polystyrene and polyvinyltoluene latex) or biological markers (autoclaved yeast cells and glutaraldehyde-fixed goat red blood cells), or a combination of both. The specificity of PL fusion in vivo appeared to be maintained in vitro. As determined by light and electron microscopy, the fusion reaction was dependent on time and temperature, and on the initial presence of membrane around both marker particles. A minimum of 10% of the vacuoles fused by 10 min of incubation at 30 degrees C, and no rupture of the vacuoles was detected during this time. After 10 min of incubation, vacuole rupture began and fusion ceased. At a constant initial vacuole concentration, the extent of PL fusion in vitro was quantitatively reproducible. This appears to be a promising system for further investigation of membrane fusion in the lysosomal system. PMID:1245550

  6. Efficient activation of human T cells of both CD4 and CD8 subsets by urease-deficient recombinant Mycobacterium bovis BCG that produced a heat shock protein 70-M. tuberculosis-derived major membrane protein II fusion protein.

    PubMed

    Mukai, Tetsu; Tsukamoto, Yumiko; Maeda, Yumi; Tamura, Toshiki; Makino, Masahiko

    2014-01-01

    For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8(+) T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8(+) T cells and perforin-producing effector CD8(+) T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

  7. Induction of cross-priming of naive CD8+ T lymphocytes by recombinant bacillus Calmette-Guerin that secretes heat shock protein 70-major membrane protein-II fusion protein.

    PubMed

    Mukai, Tetsu; Maeda, Yumi; Tamura, Toshiki; Matsuoka, Masanori; Tsukamoto, Yumiko; Makino, Masahiko

    2009-11-15

    Because Mycobacterium bovis bacillus Calmette-Guérin (BCG) unconvincingly activates human naive CD8(+) T cells, a rBCG (BCG-70M) that secretes a fusion protein comprising BCG-derived heat shock protein (HSP)70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed to potentiate the ability of activating naive CD8(+) T cells through dendritic cells (DC). BCG-70M secreted HSP70-MMP-II fusion protein in vitro, which stimulated DC to produce IL-12p70 through TLR2. BCG-70M-infected DC activated not only memory and naive CD8(+) T cells, but also CD4(+) T cells of both types to produce IFN-gamma. The activation of these naive T cells by BCG-70M was dependent on the MHC and CD86 molecules on BCG-70M-infected DC, and was significantly inhibited by pretreatment of DC with chloroquine. Both brefeldin A and lactacystin significantly inhibited the activation of naive CD8(+) T cells by BCG-70M through DC. Thus, the CD8(+) T cell activation may be induced by cross-presentation of Ags through a TAP- and proteosome-dependent cytosolic pathway. When naive CD8(+) T cells were stimulated by BCG-70M-infected DC in the presence of naive CD4(+) T cells, CD62L(low)CD8(+) T cells and perforin-producing CD8(+) T cells were efficiently produced. MMP-II-reactive CD4(+) and CD8(+) memory T cells were efficiently produced in C57BL/6 mice by infection with BCG-70M. These results indicate that BCG-70M activated DC, CD4(+) T cells, and CD8(+) T cells, and the combination of HSP70 and MMP-II may be useful for inducing better T cell activation.

  8. Fusion Power.

    ERIC Educational Resources Information Center

    Dingee, David A.

    1979-01-01

    Discusses the extraordinary potential, the technical difficulties, and the financial problems that are associated with research and development of fusion power plants as a major source of energy. (GA)

  9. Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

    SciTech Connect

    Petit, Chad M.; Chouljenko, Vladimir N.; Iyer, Arun; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G. . E-mail: vtgusk@lsu.edu

    2007-04-10

    The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of {sup 3}H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion.

  10. Full Inactivation of Human Influenza Virus by High Hydrostatic Pressure Preserves Virus Structure and Membrane Fusion While Conferring Protection to Mice against Infection

    PubMed Central

    Dumard, Carlos H.; Barroso, Shana P. C.; de Oliveira, Guilherme A. P.; Carvalho, Carlos A. M.; Gomes, Andre M. O.; Couceiro, José Nelson S. S.; Ferreira, Davis F.; Nico, Dirlei; Oliveira, Andrea C.; Silva, Jerson L.; Santos, Patrícia S.

    2013-01-01

    Whole inactivated vaccines (WIVs) possess greater immunogenicity than split or subunit vaccines, and recent studies have demonstrated that WIVs with preserved fusogenic activity are more protective than non-fusogenic WIVs. In this work, we describe the inactivation of human influenza virus X-31 by high hydrostatic pressure (HHP) and analyze the effects on the structure by spectroscopic measurements, light scattering, and electron microscopy. We also investigated the effects of HHP on the glycoprotein activity and fusogenic activity of the viral particles. The electron microscopy data showed pore formation on the viral envelope, but the general morphology was preserved, and small variations were seen in the particle structure. The activity of hemagglutinin (HA) during the process of binding and fusion was affected in a time-dependent manner, but neuraminidase (NA) activity was not affected. Infectious activity ceased after 3 hours of pressurization, and mice were protected from infection after being vaccinated. Our results revealed full viral inactivation with overall preservation of viral structure and maintenance of fusogenic activity, thereby conferring protection against infection. A strong response consisting of serum immunoglobulin IgG1, IgG2a, and serum and mucosal IgA was also detected after vaccination. Thus, our data strongly suggest that applying hydrostatic pressure may be an effective method for developing new vaccines against influenza A as well as other viruses. PMID:24282553

  11. Surface aggregation patterns of LDL receptors near coated pits III: potential effects of combined retrograde membrane flow-diffusion and a polarized-insertion mechanism.

    PubMed

    Echavarria-Heras, Héctor; Leal-Ramirez, Cecilia; Castillo, Oscar

    2014-05-22

    Although the process of endocytosis of the low density lipoprotein (LDL) macromolecule and its receptor have been the subject of intense experimental research and modeling, there are still conflicting hypotheses and even conflicting data regarding the way receptors are transported to coated pits, the manner by which receptors are inserted before they aggregate in coated pits, and the display of receptors on the cell surface. At first it was considered that LDL receptors in human fibroblasts are inserted at random locations and then transported by diffusion toward coated pits. But experiments have not ruled out the possibility that the true rate of accumulation of LDL receptors in coated pits might be faster than predicted on the basis of pure diffusion and uniform reinsertion over the entire cell surface. It has been claimed that recycled LDL receptors are inserted preferentially in regions where coated pits form, with display occurring predominantly as groups of loosely associated units. Another mechanism that has been proposed by experimental cell biologists which might affect the accumulation of receptors in coated pits is a retrograde membrane flow. This is essentially linked to a polarized receptor insertion mode and also to the capping phenomenon, characterized by the formation of large patches of proteins that passively flow away from the regions of membrane exocytosis. In this contribution we calculate the mean travel time of LDL receptors to coated pits as determined by the ratio of flow strength to diffusion-coefficient, as well as by polarized-receptor insertion. We also project the resulting display of unbound receptors on the cell membrane. We found forms of polarized insertion that could potentially reduce the mean capture time of LDL receptors by coated pits which is controlled by diffusion and uniform insertion. Our results show that, in spite of its efficiency as a possible device for enhancement of the rate of receptor trapping, polarized

  12. Surface aggregation patterns of LDL receptors near coated pits III: potential effects of combined retrograde membrane flow-diffusion and a polarized-insertion mechanism

    PubMed Central

    2014-01-01

    Although the process of endocytosis of the low density lipoprotein (LDL) macromolecule and its receptor have been the subject of intense experimental research and modeling, there are still conflicting hypotheses and even conflicting data regarding the way receptors are transported to coated pits, the manner by which receptors are inserted before they aggregate in coated pits, and the display of receptors on the cell surface. At first it was considered that LDL receptors in human fibroblasts are inserted at random locations and then transported by diffusion toward coated pits. But experiments have not ruled out the possibility that the true rate of accumulation of LDL receptors in coated pits might be faster than predicted on the basis of pure diffusion and uniform reinsertion over the entire cell surface. It has been claimed that recycled LDL receptors are inserted preferentially in regions where coated pits form, with display occurring predominantly as groups of loosely associated units. Another mechanism that has been proposed by experimental cell biologists which might affect the accumulation of receptors in coated pits is a retrograde membrane flow. This is essentially linked to a polarized receptor insertion mode and also to the capping phenomenon, characterized by the formation of large patches of proteins that passively flow away from the regions of membrane exocytosis. In this contribution we calculate the mean travel time of LDL receptors to coated pits as determined by the ratio of flow strength to diffusion-coefficient, as well as by polarized-receptor insertion. We also project the resulting display of unbound receptors on the cell membrane. We found forms of polarized insertion that could potentially reduce the mean capture time of LDL receptors by coated pits which is controlled by diffusion and uniform insertion. Our results show that, in spite of its efficiency as a possible device for enhancement of the rate of receptor trapping, polarized

  13. Separation and determination of metallocyanide complexes of Fe(II), Ni(II) and Co(III) by ion-interaction chromatography with membrane suppressed conductivity detection applied to analysis of oil refinery streams (sour water).

    PubMed

    Souza e Silva, Renata; de Carvalho, Maria de Fátima Batista; Santelli, Ricardo Erthal

    2006-09-15

    A separation and determination method for the analysis of cyanometallic complexes of Fe(II), Ni(II) and Co(III) was developed to be applied to the analysis of petroleum refinery streams (sour water). Ion-interaction chromatography was used employing an analytical column IonPac NS1 10 microm and a chromatographic system ICS 2500 equipped with a membrane conductivity suppression ASRS ultra 4mm, both supplied by Dionex Corporation. The mobile phase was composed of 2 mmol l(-1) TBAOH, 1 mmol l(-1) Na(2)CO(3), 0.1 mol l(-1) NaCN and ACN (77:23, v/v), flowing at 0.7 ml min(-1). At the optimized conditions, detection limits estimated by the calibration curve parameters and relative standard deviation were: 0.002 mg CNl(-1) and 3.1% for Fe(CN)(6)(4-); 0.003 mg CNl(-1) and 2.5% for Ni(CN)(4)(2-) and 0.003 mg CNl(-1) and 2.8% for Co(CN)(6)(3-). Sour water samples without any pretreatment (except membrane filtration) from a petroleum refinery in Brazil were analyzed successfully by external calibration method. PMID:16889783

  14. Rho GTPase activity modulates paramyxovirus fusion protein-mediated cell-cell fusion

    SciTech Connect

    Schowalter, Rachel M.; Wurth, Mark A.; Aguilar, Hector C.; Lee, Benhur; Moncman, Carole L.; McCann, Richard O.; Dutch, Rebecca Ellis . E-mail: rdutc2@uky.edu

    2006-07-05

    The paramyxovirus fusion protein (F) promotes fusion of the viral envelope with the plasma membrane of target cells as well as cell-cell fusion. The plasma membrane is closely associated with the actin cytoskeleton, but the role of actin dynamics in paramyxovirus F-mediated membrane fusion is unclear. We examined cell-cell fusion promoted by two different paramyxovirus F proteins in three cell types in the presence of constitutively active Rho family GTPases, major cellular coordinators of actin dynamics. Reporter gene and syncytia assays demonstrated that expression of either Rac1{sup V12} or Cdc42{sup V12} could increase cell-cell fusion promoted by the Hendra or SV5 glycoproteins, though the effect was dependent on the cell type expressing the viral glycoproteins. In contrast, RhoA{sup L63} decreased cell-cell fusion promoted by Hendra glycoproteins but had little affect on SV5 F-mediated fusion. Also, data suggested that GTPase activation in the viral glycoprotein-containing cell was primarily responsible for changes in fusion. Additionally, we found that activated Cdc42 promoted nuclear rearrangement in syncytia.

  15. Broad-spectrum antivirals against viral fusion

    PubMed Central

    Vigant, Frederic; Santos, Nuno C.; Lee, Benhur

    2015-01-01

    Effective antivirals have been developed against specific viruses, such as HIV, Hepatitis C virus and influenza virus. This ‘one bug–one drug’ approach to antiviral drug development can be successful, but it may be inadequate for responding to an increasing diversity of viruses that cause significant diseases in humans. The majority of viral pathogens that cause emerging and re-emerging infectious diseases are membrane-enveloped viruses, which require the fusion of viral and cell membranes for virus entry. Therefore, antivirals that target the membrane fusion process represent new paradigms for broad-spectrum antiviral discovery. In this Review, we discuss the mechanisms responsible for the fusion between virus and cell membranes and explore how broad-spectrum antivirals target this process to prevent virus entry. PMID:26075364

  16. Laser fusion

    SciTech Connect

    Smit, W.A.; Boskma, P.

    1980-12-01

    Unrestricted laser fusion offers nations an opportunity to circumvent arms control agreements and develop thermonuclear weapons. Early laser weapons research sought a clean radiation-free bomb to replace the fission bomb, but this was deceptive because a fission bomb was needed to trigger the fusion reaction and additional radioactivity was induced by generating fast neutrons. As laser-implosion experiments focused on weapons physics, simulating weapons effects, and applications for new weapons, the military interest shifted from developing a laser-ignited hydrogen bomb to more sophisticated weapons and civilian applications for power generation. Civilian and military research now overlap, making it possible for several countries to continue weapons activities and permitting proliferation of nuclear weapons. These countries are reluctant to include inertial confinement fusion research in the Non-Proliferation Treaty. 16 references. (DCK)

  17. Membranes in motion

    PubMed Central

    van Meer, Gerrit

    2010-01-01

    The Keystone Symposium on the Molecular Basis for Biological Membrane Organization and Dynamics held in January this year offered new insights into the molecular machines at work in cells. Topics included the machinery responsible for the dynamic shape of organelles, the budding and fusion of vesicular carriers, and the intricate sorting systems that ensure the correct delivery of cellular components. PMID:20395954

  18. Fusion pore regulation of transmitter release.

    PubMed

    Fernández-Peruchena, Carlos; Navas, Sergio; Montes, María A; Alvarez de Toledo, Guillermo

    2005-09-01

    During the last decade a wealth of new information about the properties of the exocytotic fusion pore is changing our current view of exocytosis. The exocytotic fusion pore, a necessary stage before the full merging of the vesicle membrane with the plasma membrane, is becoming a key cellular structure that might critically control the amount of neurotransmitter released into the synaptic cleft and that can be subjected to control by second messengers and phosphorylated proteins. Fusion pores form, expand to fully merge membranes, or can close leaving an intact and identical synaptic vesicle in place for a new round of exocytosis. Transient formation of fusion pores is the mechanistic representation of the "kiss-and-run" hypothesis of transmitter release and offers new alternatives for synaptic vesicle recycling besides to the classical mechanism mediated by clathrin coat endocytosis. For vesicle recycling transient fusion pores ensures a fast mechanism for maintaining an active pool of synaptic vesicles. The size reached by transient fusion pores and the time spent on the open state can determine the release of subquantal synaptic transmission, which could be a mechanism of synaptic potentiation. In this review we will described the electrophysiological and fluorescence methods that contribute to further explore the biophysical properties of the exocytotic fusion pore and the relevant experiments obtained by these methods.

  19. Human keratinocytes restrict chikungunya virus replication at a post-fusion step.

    PubMed

    Bernard, Eric; Hamel, Rodolphe; Neyret, Aymeric; Ekchariyawat, Peeraya; Molès, Jean-Pierre; Simmons, Graham; Chazal, Nathalie; Desprès, Philippe; Missé, Dorothée; Briant, Laurence

    2015-02-01

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses.

  20. SAGE III

    Atmospheric Science Data Center

    2016-06-15

    SAGE III Data and Information The Stratospheric Aerosol and Gas ... on the spacecraft. SAGE III produced L1 and L2 scientific data from 5/07/2002 until 12/31/2005. The flight of the second instrument is as ... Guide Documents:  Project Guide Data Products User's Guide  (PDF) Relevant Documents:  ...

  1. Binding of [3H](2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl) glycine ([3H]LY341495) to cell membranes expressing recombinant human group III metabotropic glutamate receptor subtypes.

    PubMed

    Wright, R A; Arnold, M B; Wheeler, W J; Ornstein, P L; Schoepp, D D

    2000-12-01

    LY341495 is a highly potent and selective antagonist for group II mGlu receptors (mGlu2 and mGlu3). High affinity binding of [3H]LY341495 to recombinant human group II mGlu receptors (mGlu2 and mGlu3), and in rat brain homogenates (Kd approximately 1 nM), has been previously described. Although LY341495 is a very selective nM-potent antagonist for group II mGlu receptors, it is also a relatively potent antagonist for group III mGlu receptors at high nanomolar to low micromolar concentrations. In this study we examined and characterized the binding of [3H]LY341495 to membranes of cells expressing recombinant human group III mGlu receptors. Using up to 100 nM of [3H]LY341495, the level of specific binding in human mGlu4a receptor-expressing cell membranes was not appreciable and binding to this site was not examined further. In contrast, we demonstrated sufficient specific binding of [3H]LY341495 to human mGlu6, mGlu7a and mGlu8a receptor-expressing cell membranes to allow for further characterizations. [3H]LY341495 binding was saturable and rapidly reversible. [3H]LY341495 bound to a single site in each cell line, with Kd and Bmax values of 31.6+/-6.8 nM and 3.3+/-0.7 pmol/mg protein (mGlu6), 72.7+/-22.0 nM and 3.7+/-0.4 pmol/mg protein (mGlu7a), and 14.0+/-1.1 nM and 3.0+/-0.2 pmol/mg protein (mGlu8a). [3H]LY341495 binding to mGlu6, 7a and 8a was displaceable by compounds which interact functionally with group III mGlu receptors. For example, L-AP4 displaced [3H]LY341495 with Ki values of 6.8+/-3.1 microM (mGlu6), 211+/-43 microM (mGlu7a) and 1.6+/-0.3 microM (mGlu8a). With L-glutamate, we obtained Ki values of 12.3+/-3.5, 869+/-154 and 4.5+/-0.83 microM, for mGlu6, mGlu7a and mGlu8a, respectively. Ki values for unlabelled LY341495 were 0.058+/-0.008, 0.22+/-0.05 and 0.029+/-0.008 microM, respectively. These studies demonstrated that [3H]LY341495 is a useful radioligand for studying the pharmacology and expression of recombinant mGlu6, 7a and 8a receptors in cell

  2. Reduction of Influenza Virus Envelope's Fusogenicity by Viral Fusion Inhibitors.

    PubMed

    Rowse, Michael; Qiu, Shihong; Tsao, Jun; Yamauchi, Yohei; Wang, Guoxin; Luo, Ming

    2016-01-01

    During cell entry of an enveloped virus, the viral membrane must be fused with the cellular membrane. The virus envelope has a unique structure consisting of viral proteins and a virus-specific lipid composition, whereas the host membrane has its own structure with host membrane proteins. Compound 136 was previously found to bind in close proximity to the viral envelope and inhibit influenza virus entry. We showed here that the 136-treated influenza virus still caused hemolysis. When liposomes were used as the target membrane for 136-treated viruses, aberrant fusion occurred; few liposomes fused per virion, and glycoproteins were not distributed evenly across fusion complexes. Additionally, large fusion aggregates did not form, and in some instances, neck-like structures were found. Based on previous results and hemolysis, fusion inhibition by 136 occurs post-scission but prior to lipid mixing. PMID:27622947

  3. Cold fusion, Alchemist's dream

    SciTech Connect

    Clayton, E.D.

    1989-09-01

    In this report the following topics relating to cold fusion are discussed: muon catalysed cold fusion; piezonuclear fusion; sundry explanations pertaining to cold fusion; cosmic ray muon catalysed cold fusion; vibrational mechanisms in excited states of D{sub 2} molecules; barrier penetration probabilities within the hydrogenated metal lattice/piezonuclear fusion; branching ratios of D{sub 2} fusion at low energies; fusion of deuterons into {sup 4}He; secondary D+T fusion within the hydrogenated metal lattice; {sup 3}He to {sup 4}He ratio within the metal lattice; shock induced fusion; and anomalously high isotopic ratios of {sup 3}He/{sup 4}He.

  4. Efforts in Public Relations on Fusion in Europe

    NASA Astrophysics Data System (ADS)

    Ongena, J.; van Oost, G.

    2001-10-01

    An overview will be given of different published materials currently in use in Europe for public relations on fusion. We will also present a CD-ROM for individual and classroom use, containing (i) a general background on different energy forms, (ii) general principles of fusion, (iii) current research efforts and (iv) future prospects of fusion. This CD-ROM is currently in English, German, French, Spanish, Portuguese and Italian. Fusion posters developed in collaboration with CPEP in Dutch, French, German, Italian, Spanish and Portuguese will be shown. Several new brochures and leaflets intended to increase the public awareness on fusion in Europe will be on display.

  5. Restricted movement of lipid and aqueous dyes through pores formed by influenza hemagglutinin during cell fusion

    PubMed Central

    1994-01-01

    The fusion of cells by influenza hemagglutinin (HA) is the best characterized example of protein-mediated membrane fusion. In simultaneous measurements of pairs of assays for fusion, we determined the order of detectable events during fusion. Fusion pore formation in HA-triggered cell-cell fusion was first detected by changes in cell membrane capacitance, next by a flux of fluorescent lipid, and finally by flux of aqueous fluorescent dye. Fusion pore conductance increased by small steps. A retardation of lipid and aqueous dyes occurred during fusion pore fluctuations. The flux of aqueous dye depended on the size of the molecule. The lack of movement of aqueous dyes while total fusion pore conductance increased suggests that initial HA-triggered fusion events are characterized by the opening of multiple small pores: the formation of a "sieve". PMID:7806567

  6. Evaluation of Salmonella enterica type III secretion system effector proteins as carriers for heterologous vaccine antigens.

    PubMed

    Hegazy, Wael Abdel Halim; Xu, Xin; Metelitsa, Leonid; Hensel, Michael

    2012-03-01

    Live attenuated strains of Salmonella enterica have a high potential as carriers of recombinant vaccines. The type III secretion system (T3SS)-dependent translocation of S. enterica can be deployed for delivery of heterologous antigens to antigen-presenting cells. Here we investigated the efficacy of various effector proteins of the Salmonella pathogenicity island (SPI2)-encoded T3SS for the translocation of model antigens and elicitation of immune responses. The SPI2 T3SS effector proteins SifA, SteC, SseL, SseJ, and SseF share an endosomal membrane-associated subcellular localization after translocation. We observed that all effector proteins could be used to translocate fusion proteins with the model antigens ovalbumin and listeriolysin into the cytosol of host cells. Under in vitro conditions, fusion proteins with SseJ and SteC stimulated T-cell responses that were superior to those triggered by fusion proteins with SseF. However, in mice vaccinated with Salmonella carrier strains, only fusion proteins based on SseJ or SifA elicited potent T-cell responses. These data demonstrate that the selection of an optimal SPI2 effector protein for T3SS-mediated translocation is a critical parameter for the rational design of effective Salmonella-based recombinant vaccines.

  7. Public Relations on Fusion in Europe

    NASA Astrophysics Data System (ADS)

    Ongena, J.; van Oost, G.; Paris, P. J.

    2000-10-01

    A summary will be presented of PR efforts on fusion energy research in Europe. A 3-D movie of a fusion research experimental reactor has been realized at the start of this year. It has been made entirely on virtual animation basis. Two versions exists, a short version of 3 min., as a video clip, and a longer version of nearly 8 min. Both could be viewed in 3D, using special projections and passive glasses or in normal VHS video projections. A new CD-ROM for individual and classroom use will be presented, discussing (i) the different energy forms, (ii) general principles of fusion, (iii) current research efforts and (iv) future prospects of fusion. This CD-ROM is now produced in English, German, French, Spanish, Italian and Portuguese Several new brochures and leaflets intended to increase the public awareness on fusion in Europe will be on display.

  8. Neutron diffraction studies of viral fusion peptides

    NASA Astrophysics Data System (ADS)

    Bradshaw, Jeremy P.; J. M. Darkes, Malcolm; Katsaras, John; Epand, Richard M.

    2000-03-01

    Membrane fusion plays a vital role in a large and diverse number of essential biological processes. Despite this fact, the precise molecular events that occur during fusion are still not known. We are currently engaged on a study of membrane fusion as mediated by viral fusion peptides. These peptides are the N-terminal regions of certain viral envelope proteins that mediate the process of fusion between the viral envelope and the membranes of the host cell during the infection process. As part of this study, we have carried out neutron diffraction measurements at the ILL, BeNSC and Chalk River, on a range of viral fusion peptides. The peptides, from simian immunodeficiency virus (SIV), influenza A and feline leukaemia virus (FeLV), were incorporated into stacked phospholipid bilayers. Some of the peptides had been specifically deuterated at key amino acids. Lamellar diffraction data were collected and analysed to yield information on the peptide conformation, location and orientation relative to the bilayer.

  9. Late stages of the synchronized macrophage fusion in osteoclast formation depend on dynamin.

    PubMed

    Verma, Santosh K; Leikina, Evgenia; Melikov, Kamran; Chernomordik, Leonid V

    2014-12-15

    Macrophage fusion that leads to osteoclast formation is one of the most important examples of cell-cell fusion in development, tissue homoeostasis and immune response. Protein machinery that fuses macrophages remains to be identified. In the present study, we explored the fusion stage of osteoclast formation for RAW macrophage-like murine cells and for macrophages derived from human monocytes. To uncouple fusion from the preceding differentiation processes, we accumulated fusion-committed cells in the presence of LPC (lysophosphatidylcholine) that reversibly blocks membrane merger. After 16 h, we removed LPC and observed cell fusion events that would normally develop within 16 h develop instead within 30-90 min. Thus, whereas osteoclastogenesis, generally, takes several days, our approach allowed us to focus on an hour in which we observe robust fusion between the cells. Complementing syncytium formation assay with a novel membrane merger assay let us study the synchronized fusion events downstream of a local merger between two plasma membranes, but before expansion of nascent membrane connections and complete unification of the cells. We found that the expansion of membrane connections detected as a growth of multinucleated osteoclasts depends on dynamin activity. In contrast, a merger between the plasma membranes of the two cells was not affected by inhibitors of dynamin GTPase. Thus dynamin that was recently found to control late stages of myoblast fusion also controls late stages of macrophage fusion, revealing an intriguing conserved mechanistic motif shared by diverse cell-cell fusion processes. PMID:25336256