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Sample records for membrane fusion iii

  1. The interaction of alphavirus E1 protein with exogenous domain III defines stages in virus-membrane fusion.

    PubMed

    Roman-Sosa, Gleyder; Kielian, Margaret

    2011-12-01

    Alphaviruses such as Semliki Forest virus (SFV) are enveloped viruses that infect cells through a low-pH-triggered membrane fusion reaction mediated by the transmembrane fusion protein E1. E1 drives fusion by insertion of its hydrophobic fusion loop into the cell membrane and refolding to a stable trimeric hairpin. In this postfusion conformation, the immunoglobulin-like domain III (DIII) and the stem region pack against the central core of the trimer. Membrane fusion and infection can be specifically inhibited by exogenous DIII, which binds to an intermediate in the E1 refolding pathway. Here we characterized the properties of the E1 target for interaction with exogenous DIII. The earliest target for DIII binding was an extended membrane-inserted E1 trimer, which was not detectable by assays for the stable postfusion hairpin. DIII binding provided a tool to detect this extended trimer and to define a series of SFV fusion-block mutants. DIII binding studies showed that the mutants were blocked in distinct steps in fusion protein refolding. Our results suggested that formation of the initial extended trimer was reversible and that it was stabilized by the progressive fold-back of the DIII and stem regions.

  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

    PubMed Central

    Harrison, Stephen C.

    2015-01-01

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. PMID:25866377

  4. Fusion Power Demonstration III

    SciTech Connect

    Lee, J.D.

    1985-07-01

    This is the third in the series of reports covering the Fusion Power Demonstration (FPD) design study. This volume considers the FPD-III configuration that incorporates an octopole end plug. As compared with the quadrupole end-plugged designs of FPD-I and FPD-II, this octopole configuration reduces the number of end cell magnets and shortens the minimum ignition length of the central cell. The end-cell plasma length is also reduced, which in turn reduces the size and cost of the end cell magnets and shielding. As a contiuation in the series of documents covering the FPD, this report does not stand alone as a design description of FPD-III. Design details of FPD-III subsystems that do not differ significantly from those of the FPD-II configuration are not duplicated in this report.

  5. 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.

  6. Viral membrane fusion

    PubMed Central

    Harrison, Stephen C

    2008-01-01

    Infection by viruses having lipid-bilayer envelopes proceeds through fusion of the viral membrane with a membrane of the target cell. Viral ‘fusion proteins’ facilitate this process. They vary greatly in structure, but all seem to have a common mechanism of action, in which a ligand-triggered, large-scale conformational change in the fusion protein is coupled to apposition and merger of the two bilayers. We describe three examples—the influenza virus hemagglutinin, the flavivirus E protein and the vesicular stomatitis virus G protein—in some detail, to illustrate the ways in which different structures have evolved to implement this common mechanism. Fusion inhibitors can be effective antiviral agents. PMID:18596815

  7. Membrane fusion during poxvirus entry.

    PubMed

    Moss, Bernard

    2016-12-01

    Poxviruses comprise a large family of enveloped DNA viruses that infect vertebrates and invertebrates. Poxviruses, unlike most DNA viruses, replicate in the cytoplasm and encode enzymes and other proteins that enable entry, gene expression, genome replication, virion assembly and resistance to host defenses. Entry of vaccinia virus, the prototype member of the family, can occur at the plasma membrane or following endocytosis. Whereas many viruses encode one or two proteins for attachment and membrane fusion, vaccinia virus encodes four proteins for attachment and eleven more for membrane fusion and core entry. The entry-fusion proteins are conserved in all poxviruses and form a complex, known as the Entry Fusion Complex (EFC), which is embedded in the membrane of the mature virion. An additional membrane that encloses the mature virion and is discarded prior to entry is present on an extracellular form of the virus. The EFC is held together by multiple interactions that depend on nine of the eleven proteins. The entry process can be divided into attachment, hemifusion and core entry. All eleven EFC proteins are required for core entry and at least eight for hemifusion. To mediate fusion the virus particle is activated by low pH, which removes one or more fusion repressors that interact with EFC components. Additional EFC-interacting fusion repressors insert into cell membranes and prevent secondary infection. The absence of detailed structural information, except for two attachment proteins and one EFC protein, is delaying efforts to determine the fusion mechanism.

  8. 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.

  9. Membrane fusion in muscle development and repair

    PubMed Central

    Demonbreun, Alexis R.; Biersmith, Bridget H.

    2015-01-01

    Mature skeletal muscle forms from the fusion of skeletal muscle precursor cells, myoblasts. Myoblasts fuse to other myoblasts to generate multinucleate myotubes during myogenesis, and myoblasts also fuse to other myotubes during muscle growth and repair. Proteins within myoblasts and myotubes regulate complex processes such as elongation, migration, cell adherence, cytoskeletal reorganization, membrane coalescence, and ultimately fusion. Recent studies have identified cell surface proteins, intracellular proteins, and extracellular signaling molecules required for the proper fusion of muscle. Many proteins that actively participate in myoblast fusion also coordinate membrane repair. Here we will review mammalian membrane fusion with specific attention to proteins that mediate myoblast fusion and muscle repair. PMID:26537430

  10. Mechanisms of influenza viral membrane fusion.

    PubMed

    Blijleven, Jelle S; Boonstra, Sander; Onck, Patrick R; van der Giessen, Erik; van Oijen, Antoine M

    2016-12-01

    Influenza viral particles are enveloped by a lipid bilayer. A major step in infection is fusion of the viral and host cellular membranes, a process with large kinetic barriers. Influenza membrane fusion is catalyzed by hemagglutinin (HA), a class I viral fusion protein activated by low pH. The exact nature of the HA conformational changes that deliver the energy required for fusion remains poorly understood. This review summarizes our current knowledge of HA structure and dynamics, describes recent single-particle experiments and modeling studies, and discusses their role in understanding how multiple HAs mediate fusion. These approaches provide a mechanistic picture in which HAs independently and stochastically insert into the target membrane, forming a cluster of HAs that is collectively able to overcome the barrier to membrane fusion. The new experimental and modeling approaches described in this review hold promise for a more complete understanding of other viral fusion systems and the protein systems responsible for cellular fusion.

  11. Photoinduced Fusion of Lipid Bilayer Membranes.

    PubMed

    Suzuki, Yui; Nagai, Ken H; Zinchenko, Anatoly; Hamada, Tsutomu

    2017-03-14

    We have developed a novel system for photocontrol of the fusion of lipid vesicles through the use of a photosensitive surfactant containing an azobenzene moiety (AzoTAB). Real-time microscopic observations clarified a change in both the surface area and internal volume of vesicles during fusion. We also determined the optimal cholesterol concentrations and temperature for inducing fusion. The mechanism of fusion can be attributed to a change in membrane tension, which is caused by the solubilization of lipids through the isomerization of AzoTAB. We used a micropipet technique to estimate membrane tension and discuss the mechanism of fusion in terms of membrane elastic energy. The obtained results regarding this novel photoinduced fusion could lead to a better understanding of the mechanism of membrane fusion in living cells and may also see wider applications, such as in drug delivery and biomimetic material design.

  12. Henipavirus membrane fusion and viral entry.

    PubMed

    Aguilar, Hector C; Iorio, Ronald M

    2012-01-01

    Nipah (NiV) and Hendra (HeV) viruses cause cell-cell fusion (syncytia) in brain, lung, heart, and kidney tissues, leading to encephalitis, pneumonia, and often death. Membrane fusion is essential to both viral entry and virus-induced cell-cell fusion, a hallmark of henipavirus infections. Elucidiation of the mechanism(s) of membrane fusion is critical to understanding henipavirus pathobiology and has the potential to identify novel strategies for the development of antiviral therapeutic agents. Henipavirus membrane fusion requires the coordinated actions of the viral attachment (G) and fusion (F) glycoproteins. Current henipavirus fusion models posit that attachment of NiV or HeV G to its cell surface receptors releases F from its metastable pre-fusion conformation to mediate membrane fusion. The identification of ephrinB2 and ephrinB3 as henipavirus receptors has paved the way for recent advances in our understanding of henipavirus membrane fusion. These advances highlight mechanistic similarities and differences between membrane fusion for the henipavirus and other genera within the Paramyxoviridae family. Here, we review these mechanisms and the current gaps in our knowledge in the field.

  13. Mechanical tension drives cell membrane fusion.

    PubMed

    Kim, Ji Hoon; Ren, Yixin; Ng, Win Pin; Li, Shuo; Son, Sungmin; Kee, Yee-Seir; Zhang, Shiliang; Zhang, Guofeng; Fletcher, Daniel A; Robinson, Douglas N; Chen, Elizabeth H

    2015-03-09

    Membrane fusion is an energy-consuming process that requires tight juxtaposition of two lipid bilayers. Little is known about how cells overcome energy barriers to bring their membranes together for fusion. Previously, we have shown that cell-cell fusion is an asymmetric process in which an "attacking" cell drills finger-like protrusions into the "receiving" cell to promote cell fusion. Here, we show that the receiving cell mounts a Myosin II (MyoII)-mediated mechanosensory response to its invasive fusion partner. MyoII acts as a mechanosensor, which directs its force-induced recruitment to the fusion site, and the mechanosensory response of MyoII is amplified by chemical signaling initiated by cell adhesion molecules. The accumulated MyoII, in turn, increases cortical tension and promotes fusion pore formation. We propose that the protrusive and resisting forces from fusion partners put the fusogenic synapse under high mechanical tension, which helps to overcome energy barriers for membrane apposition and drives cell membrane fusion.

  14. 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.

    PubMed

    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 72h post-infection, suggesting that the replication block occurred prior to virus-mediated membrane fusion.

  15. 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.

  16. 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.

  17. 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

  18. Sequential conformational rearrangements in flavivirus membrane fusion

    PubMed Central

    Chao, Luke H; Klein, Daryl E; Schmidt, Aaron G; Peña, Jennifer M; Harrison, Stephen C

    2014-01-01

    The West Nile Virus (WNV) envelope protein, E, promotes membrane fusion during viral cell entry by undergoing a low-pH triggered conformational reorganization. We have examined the mechanism of WNV fusion and sought evidence for potential intermediates during the conformational transition by following hemifusion of WNV virus-like particles (VLPs) in a single particle format. We have introduced specific mutations into E, to relate their influence on fusion kinetics to structural features of the protein. At the level of individual E subunits, trimer formation and membrane engagement of the threefold clustered fusion loops are rate-limiting. Hemifusion requires at least two adjacent trimers. Simulation of the kinetics indicates that availability of competent monomers within the contact zone between virus and target membrane makes trimerization a bottleneck in hemifusion. We discuss the implications of the model we have derived for mechanisms of membrane fusion in other contexts. DOI: http://dx.doi.org/10.7554/eLife.04389.001 PMID:25479384

  19. Cholesterol Mediates Membrane Curvature during Fusion Events

    NASA Astrophysics Data System (ADS)

    Ivankin, Andrey; Kuzmenko, Ivan; Gidalevitz, David

    2012-06-01

    Biomembranes undergo extensive shape changes as they perform vital cellular functions. The mechanisms by which lipids and proteins control membrane curvature remain unclear. We use x-ray reflectivity, grazing incidence x-ray diffraction, and epifluorescence microscopy to study binding of HIV-1 glycoprotein gp41’s membrane-bending domain to DPPC/cholesterol monolayers of various compositions at the air-liquid interface. The results offer a new insight into how membrane curvature could be regulated by cholesterol during fusion of the viral lipid envelope and the host cell membranes.

  20. Intermediates and kinetics of membrane fusion.

    PubMed Central

    Bentz, J

    1992-01-01

    Recently, it has become clear that the influenza virus fusion protein, hemagglutinin (HA), produces membrane destabilization and fusion by a multistep process, which involves the aggregation of the HAs to form a fusion site. While the details of this process are under debate, it is important to recognize that proposing any sequence of "microscopic" fusion intermediates encumbers general "macroscopic" kinetic consequences, i.e., with respect to membrane mixing rates. Using a kinetic scheme which incorporates the essential elements of several recently proposed models, some of these measurable properties have been elucidated. First, a rigorous mathematical relationship between fusion intermediates and the fusion event itself is defined. Second, it is shown that what is measured as the macroscopic "fusion rate constant" is a simple function of all of the rate constants governing the transitions between intermediates, whether or not one of the microscopic steps is rate limiting. Third, while this kinetic scheme predicts a delay (or lag) time for fusion, as has been observed, it will be very difficult to extract reliable microscopic information from these data. Furthermore, it is predicted that the delay time can depend upon HA surface density even when the HA aggregation step is very rapid compared with fusion, i.e., the delay time need not be due to HA aggregation. Fourth, the inactivation process observed for influenza virions at low pH can be described within this kinetic scheme simply, yet rigorously, via the loss of the fusion intermediates. Fifth, predicted Arrhenius plots of fusion rates can be linear for this multistep scheme, even though there is no single rate-determining step and even when a branched step is introduced, i.e., where one pathway predominates at low temperature and the other pathway predominates at high temperature. Furthermore, the apparent activation energies obtained from these plots bear little or no quantitative resemblance to the

  1. Functional relevance of transmembrane domains in membrane fusion.

    PubMed

    Nikolaus, Jörg; Herrmann, Andreas

    2012-11-01

    Membrane fusion is ubiquitous in life. Fusion of biological membranes is mediated by specialized fusion proteins anchored to the bilayers destined to fuse. Here we describe these proteins as being instrumental in viral, intracellular and developmental fusion. Next, we review experimental and theoretical evidence that points to fusion in the different systems as following a common 'fusion through hemifusion' pathway. We also focus on the structure and dynamics of the transmembrane segment that anchors the fusion proteins to the bilayer, and its role in driving fusion. In particular, we highlight the influence of this single segment on the surrounding membrane lipids and on the overall shape of the membrane along the way to fusion.

  2. Mechanism of membrane fusion induced by vesicular stomatitis virus G protein.

    PubMed

    Kim, Irene S; Jenni, Simon; Stanifer, Megan L; Roth, Eatai; Whelan, Sean P J; van Oijen, Antoine M; Harrison, Stephen C

    2017-01-03

    The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a "class I" fusogen) and West Nile virus envelope protein ("class II"). Our study of VSV now extends this description to "class III" viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion.

  3. 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

    2015-05-08

    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.

  4. Lipids as modulators of membrane fusion mediated by viral fusion proteins.

    PubMed

    Teissier, Elodie; Pécheur, Eve-Isabelle

    2007-11-01

    Enveloped viruses infect host cells by fusion of viral and target membranes. This fusion event is triggered by specific glycoproteins in the viral envelope. Fusion glycoproteins belong to either class I, class II or the newly described third class, depending upon their arrangement at the surface of the virion, their tri-dimensional structure and the location within the protein of a short stretch of hydrophobic amino acids called the fusion peptide, which is able to induce the initial lipid destabilization at the onset of fusion. Viral fusion occurs either with the plasma membrane for pH-independent viruses, or with the endosomal membranes for pH-dependent viruses. Although, viral fusion proteins are parted in three classes and the subcellular localization of fusion might vary, these proteins have to act, in common, on lipid assemblies. Lipids contribute to fusion through their physical, mechanical and/or chemical properties. Lipids can thus play a role as chemically defined entities, or through their preferential partitioning into membrane microdomains called "rafts", or by modulating the curvature of the membranes involved in the fusion process. The purpose of this review is to make a state of the art on recent findings on the contribution of cholesterol, sphingolipids and glycolipids in cell entry and membrane fusion of a number of viral families, whose members bear either class I or class II fusion proteins, or fusion proteins of the recently discovered third class.

  5. Spatial Regulation of Membrane Fusion Controlled by Modification of Phosphoinositides

    PubMed Central

    Dumas, Fabrice; Byrne, Richard D.; Vincent, Ben; Hobday, Tina M. C.; Poccia, Dominic L.; Larijani, Banafshé

    2010-01-01

    Membrane fusion plays a central role in many cell processes from vesicular transport to nuclear envelope reconstitution at mitosis but the mechanisms that underlie fusion of natural membranes are not well understood. Studies with synthetic membranes and theoretical considerations indicate that accumulation of lipids characterised by negative curvature such as diacylglycerol (DAG) facilitate fusion. However, the specific role of lipids in membrane fusion of natural membranes is not well established. Nuclear envelope (NE) assembly was used as a model for membrane fusion. A natural membrane population highly enriched in the enzyme and substrate needed to produce DAG has been isolated and is required for fusions leading to nuclear envelope formation, although it contributes only a small amount of the membrane eventually incorporated into the NE. It was postulated to initiate and regulate membrane fusion. Here we use a multidisciplinary approach including subcellular membrane purification, fluorescence spectroscopy and Förster resonance energy transfer (FRET)/two-photon fluorescence lifetime imaging microscopy (FLIM) to demonstrate that initiation of vesicle fusion arises from two unique sites where these vesicles bind to chromatin. Fusion is subsequently propagated to the endoplasmic reticulum-derived membranes that make up the bulk of the NE to ultimately enclose the chromatin. We show how initiation of multiple vesicle fusions can be controlled by localised production of DAG and propagated bidirectionally. Phospholipase C (PLCγ), GTP hydrolysis and (phosphatidylinsositol-(4,5)-bisphosphate (PtdIns(4,5)P2) are required for the latter process. We discuss the general implications of membrane fusion regulation and spatial control utilising such a mechanism. PMID:20808914

  6. Mechanism of membrane fusion induced by vesicular stomatitis virus G protein

    PubMed Central

    Kim, Irene S.; Jenni, Simon; Stanifer, Megan L.; Roth, Eatai; Whelan, Sean P. J.; van Oijen, Antoine M.; Harrison, Stephen C.

    2017-01-01

    The glycoproteins (G proteins) of vesicular stomatitis virus (VSV) and related rhabdoviruses (e.g., rabies virus) mediate both cell attachment and membrane fusion. The reversibility of their fusogenic conformational transitions differentiates them from many other low-pH-induced viral fusion proteins. We report single-virion fusion experiments, using methods developed in previous publications to probe fusion of influenza and West Nile viruses. We show that a three-stage model fits VSV single-particle fusion kinetics: (i) reversible, pH-dependent, G-protein conformational change from the known prefusion conformation to an extended, monomeric intermediate; (ii) reversible trimerization and clustering of the G-protein fusion loops, leading to an extended intermediate that inserts the fusion loops into the target-cell membrane; and (iii) folding back of a cluster of extended trimers into their postfusion conformations, bringing together the viral and cellular membranes. From simulations of the kinetic data, we conclude that the critical number of G-protein trimers required to overcome membrane resistance is 3 to 5, within a contact zone between the virus and the target membrane of 30 to 50 trimers. This sequence of conformational events is similar to those shown to describe fusion by influenza virus hemagglutinin (a “class I” fusogen) and West Nile virus envelope protein (“class II”). Our study of VSV now extends this description to “class III” viral fusion proteins, showing that reversibility of the low-pH-induced transition and architectural differences in the fusion proteins themselves do not change the basic mechanism by which they catalyze membrane fusion. PMID:27974607

  7. ROM-1 potentiates photoreceptor specific membrane fusion processes.

    PubMed

    Boesze-Battaglia, Kathleen; Stefano, Frank P; Fitzgerald, Catherine; Muller-Weeks, Susan

    2007-01-01

    Photoreceptor outer segment (OS) renewal requires a series of tightly regulated membrane fusion events which are mediated by a fusion complex containing protein and lipid components. The best characterized of these components, is a unique photoreceptor specific tetraspanin, peripherin/rds (P/rds, a.k.a., peripherin-2, Rds and Prph). In these studies we investigated the role of peripherin's non-glycosylated homolog, ROM-1, in OS fusion using a COS cell heterologous expression system and a well characterized cell free fusion assay system. Membranes isolated from COS-7 cells transfected with either FLAG-tagged P/rds or HA-tagged ROM-1 or both proteins were assayed for their ability to merge with fluorescently labeled OS plasma membrane (PM). Such membrane merger is one measure of membrane fusogenicity. The highest percent fusion was observed when the proteins were co-expressed. Furthermore detailed analysis of the fusion kinetics between fluorescently labeled PM and proteo-liposomes containing either, pure P/rds, pure ROM-1 or the ROM-1-P/rds complex clearly demonstrated that optimal fusion requires an ROM-1/P/rds complex. Proteo-liposomes composed of ROM-1 alone were not fusogenic. Peptide competition studies suggest that optimization of fusion may be due to the formation of a fusion competent peripherin/rds C-terminus in the presence of ROM-1. These studies provide further support for the hypothesis that a P/rds dependent membrane fusion complex is involved in photoreceptor renewal processes.

  8. Mutual control of membrane fission and fusion proteins.

    PubMed

    Peters, Christopher; Baars, Tonie L; Bühler, Susanne; Mayer, Andreas

    2004-11-24

    Membrane fusion and fission are antagonistic reactions controlled by different proteins. Dynamins promote membrane fission by GTP-driven changes of conformation and polymerization state, while SNAREs fuse membranes by forming complexes between t- and v-SNAREs from apposed vesicles. Here, we describe a role of the dynamin-like GTPase Vps1p in fusion of yeast vacuoles. Vps1p forms polymers that couple several t-SNAREs together. At the onset of fusion, the SNARE-activating ATPase Sec18p/NSF and the t-SNARE depolymerize Vps1p and release it from the membrane. This activity is independent of the SNARE coactivator Sec17p/alpha-SNAP and of the v-SNARE. Vps1p release liberates the t-SNAREs for initiating fusion and at the same time disrupts fission activity. We propose that reciprocal control between fusion and fission components exists, which may prevent futile cycles of fission and fusion.

  9. 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

  10. Direct observation of intermediate states in model membrane fusion

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

    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.

  11. 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

  12. The Multifaceted Role of SNARE Proteins in Membrane Fusion.

    PubMed

    Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A

    2017-01-01

    Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined.

  13. The Multifaceted Role of SNARE Proteins in Membrane Fusion

    PubMed Central

    Han, Jing; Pluhackova, Kristyna; Böckmann, Rainer A.

    2017-01-01

    Membrane fusion is a key process in all living organisms that contributes to a variety of biological processes including viral infection, cell fertilization, as well as intracellular transport, and neurotransmitter release. In particular, the various membrane-enclosed compartments in eukaryotic cells need to exchange their contents and communicate across membranes. Efficient and controllable fusion of biological membranes is known to be driven by cooperative action of SNARE proteins, which constitute the central components of the eukaryotic fusion machinery responsible for fusion of synaptic vesicles with the plasma membrane. During exocytosis, vesicle-associated v-SNARE (synaptobrevin) and target cell-associated t-SNAREs (syntaxin and SNAP-25) assemble into a core trans-SNARE complex. This complex plays a versatile role at various stages of exocytosis ranging from the priming to fusion pore formation and expansion, finally resulting in the release or exchange of the vesicle content. This review summarizes current knowledge on the intricate molecular mechanisms underlying exocytosis triggered and catalyzed by SNARE proteins. Particular attention is given to the function of the peptidic SNARE membrane anchors and the role of SNARE-lipid interactions in fusion. Moreover, the regulatory mechanisms by synaptic auxiliary proteins in SNARE-driven membrane fusion are briefly outlined. PMID:28163686

  14. Autographa californica multiple nucleopolyhedrovirus GP64 protein: Analysis of domain I and V amino acid interactions and membrane fusion activity

    SciTech Connect

    Yu, Qianlong; Blissard, Gary W.; Liu, Tong-Xian; Li, Zhaofei

    2016-01-15

    The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, but no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. - Highlights: • The baculovirus envelope glycoprotein GP64 is a class III viral fusion protein. • The detailed mechanism of conformational change of GP64 is unknown. • We analyzed 24 positions that might stabilize the post-fusion structure of GP64. • We identified 4 residues in domain V that were critical for membrane fusion. • Two residues are critical for formation of the pre-fusion conformation of GP64.

  15. Cis and trans interactions between atlastin molecules during membrane fusion

    PubMed Central

    Liu, Tina Y.; Bian, Xin; Romano, Fabian B.; Shemesh, Tom; Rapoport, Tom A.; Hu, Junjie

    2015-01-01

    Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. Here we show that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes. Tethering requires GTP hydrolysis, not just GTP binding, because the two ATL molecules are pulled together most strongly in the transition state of GTP hydrolysis. Most tethering events are futile, so that multiple rounds of GTP hydrolysis are required for successful fusion. Supported lipid bilayer experiments show that ATL molecules sitting on the same (cis) membrane can also undergo nucleotide-dependent dimerization. These results suggest that GTP hydrolysis is required to dissociate cis dimers, generating a pool of ATL monomers that can dimerize with molecules on a different (trans) membrane. In addition, tethering and fusion require the cooperation of multiple ATL molecules in each membrane. We propose a comprehensive model for ATL-mediated fusion that takes into account futile tethering and competition between cis and trans interactions. PMID:25825753

  16. Reptilian reovirus utilizes a small type III protein with an external myristylated amino terminus to mediate cell-cell fusion.

    PubMed

    Corcoran, Jennifer A; Duncan, Roy

    2004-04-01

    Reptilian reovirus is one of a limited number of nonenveloped viruses that are capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125-amino-acid fusion protein encoded by the first open reading frame of a bicistronic viral mRNA is responsible for this fusion activity. Sequence comparisons to previously characterized reovirus fusion proteins indicated that p14 represents a new member of the fusion-associated small transmembrane (FAST) protein family. Topological analysis revealed that p14 is a representative of a minor subset of integral membrane proteins, the type III proteins N(exoplasmic)/C(cytoplasmic) (N(exo)/C(cyt)), that lack a cleavable signal sequence and use an internal reverse signal-anchor sequence to direct membrane insertion and protein topology. This topology results in the unexpected, cotranslational translocation of the essential myristylated N-terminal domain of p14 across the cell membrane. The topology and structural motifs present in this novel reovirus membrane fusion protein further accentuate the diversity and unusual properties of the FAST protein family and clearly indicate that the FAST proteins represent a third distinct class of viral membrane fusion proteins.

  17. The role of the membrane-spanning domain sequence in glycoprotein-mediated membrane fusion.

    PubMed

    Taylor, G M; Sanders, D A

    1999-09-01

    The role of glycoprotein membrane-spanning domains in the process of membrane fusion is poorly understood. It has been demonstrated that replacing all or part of the membrane-spanning domain of a viral fusion protein with sequences that encode signals for glycosylphosphatidylinositol linkage attachment abrogates membrane fusion activity. It has been suggested, however, that the actual amino acid sequence of the membrane-spanning domain is not critical for the activity of viral fusion proteins. We have examined the function of Moloney murine leukemia virus envelope proteins with substitutions in the membrane-spanning domain. Envelope proteins bearing substitutions for proline 617 are processed and incorporated into virus particles normally and bind to the viral receptor. However, they possess greatly reduced or undetectable capacities for the promotion of membrane fusion and infectious virus particle formation. Our results imply a direct role for the residues in the membrane-spanning domain of the murine leukemia virus envelope protein in membrane fusion and its regulation. They also support the thesis that membrane-spanning domains possess a sequence-dependent function in other protein-mediated membrane fusion events.

  18. Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy in Chlamydomonas.

    PubMed

    Liu, Yanjie; Misamore, Michael J; Snell, William J

    2010-05-01

    The plasma membranes of gametes are specialized for fusion, yet, once fusion occurs, in many organisms the new zygote becomes incapable of further membrane fusion reactions. The molecular mechanisms that underlie this loss of fusion capacity (block to polygamy) remain unknown. During fertilization in the green alga Chlamydomonas, the plus gamete-specific membrane protein FUS1 is required for adhesion between the apically localized sites on the plasma membranes of plus and minus gametes that are specialized for fusion, and the minus-specific membrane protein HAP2 is essential for completion of the membrane fusion reaction. HAP2 (GCS1) family members are also required for fertilization in Arabidopsis, and for the membrane fusion reaction in the malaria organism Plasmodium berghei. Here, we tested whether Chlamydomonas gamete fusion triggers alterations in FUS1 and HAP2 and renders the plasma membranes of the cells incapable of subsequent fusion. We find that, even though the fusogenic sites support multi-cell adhesions, triploid zygotes are rare, indicating a fusion-triggered block to the membrane fusion reaction. Consistent with the extinction of fusogenic capacity, both FUS1 and HAP2 are degraded upon fusion. The rapid, fusion-triggered cleavage of HAP2 in zygotes is distinct from degradation occurring during constitutive turnover in gametes. Thus, gamete fusion triggers specific degradation of fusion-essential proteins and renders the zygote incapable of fusion. Our results provide the first molecular explanation for a membrane block to polygamy in any organism.

  19. Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy in Chlamydomonas

    PubMed Central

    Liu, Yanjie; Misamore, Michael J.; Snell, William J.

    2010-01-01

    The plasma membranes of gametes are specialized for fusion, yet, once fusion occurs, in many organisms the new zygote becomes incapable of further membrane fusion reactions. The molecular mechanisms that underlie this loss of fusion capacity (block to polygamy) remain unknown. During fertilization in the green alga Chlamydomonas, the plus gamete-specific membrane protein FUS1 is required for adhesion between the apically localized sites on the plasma membranes of plus and minus gametes that are specialized for fusion, and the minus-specific membrane protein HAP2 is essential for completion of the membrane fusion reaction. HAP2 (GCS1) family members are also required for fertilization in Arabidopsis, and for the membrane fusion reaction in the malaria organism Plasmodium berghei. Here, we tested whether Chlamydomonas gamete fusion triggers alterations in FUS1 and HAP2 and renders the plasma membranes of the cells incapable of subsequent fusion. We find that, even though the fusogenic sites support multi-cell adhesions, triploid zygotes are rare, indicating a fusion-triggered block to the membrane fusion reaction. Consistent with the extinction of fusogenic capacity, both FUS1 and HAP2 are degraded upon fusion. The rapid, fusion-triggered cleavage of HAP2 in zygotes is distinct from degradation occurring during constitutive turnover in gametes. Thus, gamete fusion triggers specific degradation of fusion-essential proteins and renders the zygote incapable of fusion. Our results provide the first molecular explanation for a membrane block to polygamy in any organism. PMID:20335357

  20. Temporal Control of Membrane Fusion through Photolabile PEGylation of Liposome Membranes.

    PubMed

    Kong, Li; Askes, Sven H C; Bonnet, Sylvestre; Kros, Alexander; Campbell, Frederick

    2016-01-22

    Membrane fusion results in the transport and mixing of (bio)molecules across otherwise impermeable barriers. In this communication, we describe the temporal control of targeted liposome-liposome membrane fusion and contents mixing using light as an external trigger. Our method relies on steric shielding and rapid, photoinduced deshielding of complementary fusogenic peptides tethered to opposing liposomal membranes. In an analogous approach, we were also able to demonstrate precise spatiotemporal control of liposome accumulation at cellular membranes in vitro.

  1. Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells

    PubMed Central

    Slack, J. M.; Blissard, G. W.

    2013-01-01

    Summary A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, the enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion. Spodoptera frugiperda (Sf9) insect cells expressing a chimeric Lac Repressor-IE1 protein were fused to Sf9 cells containing an EGFP reporter construct under the control of a responsive lac operator containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity. Sf9 cells expressing the Orgyia pseudotsugata Multicapsid Nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain. PMID:11562545

  2. 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.

  3. Membrane fusion mediated by coiled coils: a hypothesis.

    PubMed Central

    Bentz, J

    2000-01-01

    A molecular model of the low-pH-induced membrane fusion by influenza hemagglutinin (HA) is proposed based upon the hypothesis that the conformational change to the extended coiled coil creates a high-energy hydrophobic membrane defect in the viral envelope or HA expressing cell. It is known that 1) an aggregate of at least eight HAs is required at the fusion site, yet only two or three of these HAs need to undergo the "essential" conformational change for the first fusion pore to form (Bentz, J. 2000. Biophys. J. 78:000-000); 2) the formation of the first fusion pore signifies a stage of restricted lipid flow into the nascent fusion site; and 3) some HAs can partially insert their fusion peptides into their own viral envelopes at low pH. This suggests that the committed step for HA-mediated fusion begins with a tightly packed aggregate of HAs whose fusion peptides are inserted into their own viral envelope, which causes restricted lateral lipid flow within the HA aggregate. The transition of two or three HAs in the center of the aggregate to the extended coiled coil extracts the fusion peptide and creates a hydrophobic defect in the outer monolayer of the virion, which is stabilized by the closely packed HAs. These HAs are inhibited from diffusing away from the site to admit lateral lipid flow, in part because that would initially increase the surface area of hydrophobic exposure. The other obvious pathway to heal this hydrophobic defect, or some descendent, is recruitment of lipids from the outer monolayer of the apposed target membrane, i.e., fusion. Other viral fusion proteins and the SNARE fusion protein complex appear to fit within this hypothesis. PMID:10653801

  4. TRPM7 facilitates cholinergic vesicle fusion with the plasma membrane.

    PubMed

    Brauchi, Sebastian; Krapivinsky, Grigory; Krapivinsky, Luba; Clapham, David E

    2008-06-17

    TRPM7, of the transient receptor potential (TRP) family, is both an ion channel and a kinase. Previously, we showed that TRPM7 is located in the membranes of acetylcholine (ACh)-secreting synaptic vesicles of sympathetic neurons, forms a molecular complex with proteins of the vesicular fusion machinery, and is critical for stimulated neurotransmitter release. Here, we targeted pHluorin to small synaptic-like vesicles (SSLV) in PC12 cells and demonstrate that it can serve as a single-vesicle plasma membrane fusion reporter. In PC12 cells, as in sympathetic neurons, TRPM7 is located in ACh-secreting SSLVs. TRPM7 knockdown by siRNA, or abolishing channel activity by expression of a dominant negative TRPM7 pore mutant, decreased the frequency of spontaneous and voltage-stimulated SSLV fusion events without affecting large dense core vesicle secretion. We conclude that the conductance of TRPM7 across the vesicle membrane is important in SSLV fusion.

  5. Herpesvirus Glycoproteins Undergo Multiple Antigenic Changes before Membrane Fusion

    PubMed Central

    Glauser, Daniel L.; Kratz, Anne-Sophie; Stevenson, Philip G.

    2012-01-01

    Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4) entry machinery—gB, gH/gL and gp150—changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion. PMID:22253913

  6. Mechanics of membrane fusion/pore formation.

    PubMed

    Fuhrmans, Marc; Marelli, Giovanni; Smirnova, Yuliya G; Müller, Marcus

    2015-01-01

    Lipid bilayers play a fundamental role in many biological processes, and a considerable effort has been invested in understanding their behavior and the mechanism of topological changes like fusion and pore formation. Due to the time- and length-scale on which these processes occur, computational methods have proven to be an especially useful tool in their study. With their help, a number of interesting findings about the shape of fusion intermediates could be obtained, and novel hypotheses about the mechanism of topological changes and the involvement of peptides therein were suggested. In this work, we try to present a summary of these developments together with some hitherto unpublished results, featuring, among others, the shape of stalks and fusion pores, possible modes of action of the influenza HA fusion peptide and the SNARE protein complex, the mechanism of supported lipid bilayer formation by vesicle spreading, and the free energy and transition pathway of the fusion process.

  7. Multivalent Rab interactions determine tether-mediated membrane fusion

    PubMed Central

    Lürick, Anna; Gao, Jieqiong; Kuhlee, Anne; Yavavli, Erdal; Langemeyer, Lars; Perz, Angela; Raunser, Stefan; Ungermann, Christian

    2017-01-01

    Membrane fusion at endomembranes requires cross-talk between Rab GTPases and tethers to drive SNARE-mediated lipid bilayer mixing. Several tethers have multiple Rab-binding sites with largely untested function. Here we dissected the lysosomal HOPS complex as a tethering complex with just two binding sites for the Rab7-like Ypt7 protein to determine their relevance for fusion. Using tethering and fusion assays combined with HOPS mutants, we show that HOPS-dependent fusion requires both Rab-binding sites, with Vps39 being the stronger Ypt7 interactor than Vps41. The intrinsic amphipathic lipid packaging sensor (ALPS) motif within HOPS Vps41, a target of the vacuolar kinase Yck3, is dispensable for tethering and fusion but can affect tethering if phosphorylated. In combination, our data demonstrate that a multivalent tethering complex uses its two Rab bindings to determine the place of SNARE assembly and thus fusion at endomembranes. PMID:27852901

  8. Inner membrane fusion mediates spatial distribution of axonal mitochondria

    PubMed Central

    Yu, Yiyi; Lee, Hao-Chih; Chen, Kuan-Chieh; Suhan, Joseph; Qiu, Minhua; Ba, Qinle; Yang, Ge

    2016-01-01

    In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution. PMID:26742817

  9. Inner membrane fusion mediates spatial distribution of axonal mitochondria.

    PubMed

    Yu, Yiyi; Lee, Hao-Chih; Chen, Kuan-Chieh; Suhan, Joseph; Qiu, Minhua; Ba, Qinle; Yang, Ge

    2016-01-08

    In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution.

  10. Inner membrane fusion mediates spatial distribution of axonal mitochondria

    NASA Astrophysics Data System (ADS)

    Yu, Yiyi; Lee, Hao-Chih; Chen, Kuan-Chieh; Suhan, Joseph; Qiu, Minhua; Ba, Qinle; Yang, Ge

    2016-01-01

    In eukaryotic cells, mitochondria form a dynamic interconnected network to respond to changing needs at different subcellular locations. A fundamental yet unanswered question regarding this network is whether, and if so how, local fusion and fission of individual mitochondria affect their global distribution. To address this question, we developed high-resolution computational image analysis techniques to examine the relations between mitochondrial fusion/fission and spatial distribution within the axon of Drosophila larval neurons. We found that stationary and moving mitochondria underwent fusion and fission regularly but followed different spatial distribution patterns and exhibited different morphology. Disruption of inner membrane fusion by knockdown of dOpa1, Drosophila Optic Atrophy 1, not only increased the spatial density of stationary and moving mitochondria but also changed their spatial distributions and morphology differentially. Knockdown of dOpa1 also impaired axonal transport of mitochondria. But the changed spatial distributions of mitochondria resulted primarily from disruption of inner membrane fusion because knockdown of Milton, a mitochondrial kinesin-1 adapter, caused similar transport velocity impairment but different spatial distributions. Together, our data reveals that stationary mitochondria within the axon interconnect with moving mitochondria through fusion and fission and that local inner membrane fusion between individual mitochondria mediates their global distribution.

  11. Myomaker: A membrane activator of myoblast fusion and muscle formation

    PubMed Central

    Millay, Douglas P.; O’Rourke, Jason R.; Sutherland, Lillian B.; Bezprozvannaya, Svetlana; Shelton, John M.; Bassel-Duby, Rhonda; Olson, Eric N.

    2013-01-01

    Summary Fusion of myoblasts is essential for the formation of multi-nucleated muscle fibers. However, the identity of myogenic proteins that directly govern this fusion process has remained elusive. Here, we discovered a muscle-specific membrane protein, named Myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is down-regulated thereafter. Over-expression of Myomaker in myoblasts dramatically enhances fusion and genetic disruption of Myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibers. Remarkably, forced expression of Myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacologic perturbation of the actin cytoskeleton abolishes the activity of Myomaker, consistent with prior studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein both necessary and sufficient for mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation. PMID:23868259

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

    PubMed

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

    2014-07-29

    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.

  13. 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

  14. Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions.

    PubMed Central

    Prinz, W A; Beckwith, J

    1994-01-01

    To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions. While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not. This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm. PMID:7929016

  15. 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

  16. Vesicle-membrane fusion. Observation of simultaneous membrane incorporation and content release.

    PubMed Central

    Woodbury, D J; Hall, J E

    1988-01-01

    Vesicle fusion, the central process of neurotransmitter release and hormonal secretion, is a complex process culminating in simultaneous incorporation of vesicle membrane into the plasma membrane and release of the vesicular contents extracellularly. This report describes simultaneous observation of membrane incorporation and content release using a model system composed of a planar bilayer and dye-filled vesicles. Images FIGURE 1 PMID:2462925

  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. Oligomerization of Fusogenic Peptides Promotes Membrane Fusion by Enhancing Membrane Destabilization

    PubMed Central

    Lau, Wai Leung; Ege, David S.; Lear, James D.; Hammer, Daniel A.; DeGrado, William F.

    2004-01-01

    A key element of membrane fusion reactions in biology is the involvement of specific fusion proteins. In many viruses, the proteins that mediate membrane fusion usually exist as homotrimers. Furthermore, they contain extended triple-helical coiled-coil domains and fusogenic peptides. It has been suggested that the coiled-coil domains present the fusogenic peptide in a conformation or geometry favorable for membrane fusion. To test the hypothesis that trimerization of fusogenic peptide is related to optimal fusion, we have designed and synthesized a triple-stranded coiled-coil X31 peptide, also known as the ccX31, which mimics the influenza virus hemagglutinin fusion peptide in the fusion-active state. We compared the membrane interactive properties of ccX31 versus the monomeric X31 fusogenic peptide. Our data show that trimerization enhances peptide-induced leakage of liposomal contents and lipid mixing. Furthermore, studies using micropipette aspiration of single vesicles reveal that ccX31 decreases lysis tension, τlysis, but not area expansion modulus, Ka, of phospholipid bilayers, whereas monomeric X31 peptide lowers both τlysis and Ka. Our results are consistent with the hypothesis that oligomerization of fusogenic peptide promotes membrane fusion, possibly by enhancing localized destabilization of lipid bilayers. PMID:14695269

  19. HIV Fusion Peptide Penetrates, Disorders, and Softens T-Cell Membrane Mimics

    PubMed Central

    Tristram-Nagle, Stephanie; Chan, Rob; Kooijman, Edgar; Uppamoochikkal, Pradeep; Qiang, Wei; Weliky, David P.; Nagle, John F.

    2010-01-01

    This work investigates the interaction of N-terminal gp41 fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wild-type FP23 (23 N-terminal amino acids of gp41), (ii) water-soluble monomeric FP that adds six lysines on the C-terminus of FP23 (FPwsm), and (iii) the C-terminus covalently linked trimeric version (FPtri) of FPwsm. Model membranes were (i) LM3 (a T-cell mimic), (ii) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol, (iv) 1,2-dierucoyl-sn-glycero-3-phosphocholine, and (v) 1,2-dierucoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol. Diffuse synchrotron low-angle x-ray scattering from fully hydrated samples, supplemented by volumetric data, showed that FP23 and FPtri penetrate into the hydrocarbon region and cause membranes to thin. Depth of penetration appears to depend upon a complex combination of factors including bilayer thickness, presence of cholesterol, and electrostatics. X-ray data showed an increase in curvature in hexagonal phase 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. Low-angle x-ray scattering data also yielded the bending modulus KC, a measure of membrane stiffness, and wide-angle x-ray scattering yielded the Sxray orientational order parameter. Both FP23 and FPtri decreased KC and Sxray considerably, while the weak effect of FPwsm suggests that it did not partition strongly into LM3 model membranes. Our results are consistent with the HIV FP disordering and softening the T-cell membrane, thereby lowering the activation energy for viral membrane fusion. PMID:20655315

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. Lipid flow through fusion pores connecting membranes of different tensions.

    PubMed

    Chizmadzhev, Y A; Kumenko, D A; Kuzmin, P I; Chernomordik, L V; Zimmerberg, J; Cohen, F S

    1999-06-01

    When two membranes fuse, their components mix; this is usually described as a purely diffusional process. However, if the membranes are under different tensions, the material will spread predominantly by convection. We use standard fluid mechanics to rigorously calculate the steady-state convective flux of lipids. A fusion pore is modeled as a toroid shape, connecting two planar membranes. Each of the membrane monolayers is considered separately as incompressible viscous media with the same shear viscosity, etas. The two monolayers interact by sliding past each other, described by an intermonolayer viscosity, etar. Combining a continuity equation with an equation that balances the work provided by the tension difference, Deltasigma, against the energy dissipated by flow in the viscous membrane, yields expressions for lipid velocity, upsilon, and area of lipid flux, Phi. These expressions for upsilon and Phi depend on Deltasigma, etas, etar, and geometrical aspects of a toroidal pore, but the general features of the theory hold for any fusion pore that has a roughly hourglass shape. These expressions are readily applicable to data from any experiments that monitor movement of lipid dye between fused membranes under different tensions. Lipid velocity increases nonlinearly from a small value for small pore radii, rp, to a saturating value at large rp. As a result of velocity saturation, the flux increases linearly with pore radius for large pores. The calculated lipid flux is in agreement with available experimental data for both large and transient fusion pores.

  5. Lipid flow through fusion pores connecting membranes of different tensions.

    PubMed Central

    Chizmadzhev, Y A; Kumenko, D A; Kuzmin, P I; Chernomordik, L V; Zimmerberg, J; Cohen, F S

    1999-01-01

    When two membranes fuse, their components mix; this is usually described as a purely diffusional process. However, if the membranes are under different tensions, the material will spread predominantly by convection. We use standard fluid mechanics to rigorously calculate the steady-state convective flux of lipids. A fusion pore is modeled as a toroid shape, connecting two planar membranes. Each of the membrane monolayers is considered separately as incompressible viscous media with the same shear viscosity, etas. The two monolayers interact by sliding past each other, described by an intermonolayer viscosity, etar. Combining a continuity equation with an equation that balances the work provided by the tension difference, Deltasigma, against the energy dissipated by flow in the viscous membrane, yields expressions for lipid velocity, upsilon, and area of lipid flux, Phi. These expressions for upsilon and Phi depend on Deltasigma, etas, etar, and geometrical aspects of a toroidal pore, but the general features of the theory hold for any fusion pore that has a roughly hourglass shape. These expressions are readily applicable to data from any experiments that monitor movement of lipid dye between fused membranes under different tensions. Lipid velocity increases nonlinearly from a small value for small pore radii, rp, to a saturating value at large rp. As a result of velocity saturation, the flux increases linearly with pore radius for large pores. The calculated lipid flux is in agreement with available experimental data for both large and transient fusion pores. PMID:10354423

  6. Mechanism for Active Membrane Fusion Triggering by Morbillivirus Attachment Protein

    PubMed Central

    Ader, Nadine; Brindley, Melinda; Avila, Mislay; Örvell, Claes; Horvat, Branka; Hiltensperger, Georg; Schneider-Schaulies, Jürgen; Vandevelde, Marc; Zurbriggen, Andreas; Plemper, Richard K.

    2013-01-01

    The paramyxovirus entry machinery consists of two glycoproteins that tightly cooperate to achieve membrane fusion for cell entry: the tetrameric attachment protein (HN, H, or G, depending on the paramyxovirus genus) and the trimeric fusion protein (F). Here, we explore whether receptor-induced conformational changes within morbillivirus H proteins promote membrane fusion by a mechanism requiring the active destabilization of prefusion F or by the dissociation of prefusion F from intracellularly preformed glycoprotein complexes. To properly probe F conformations, we identified anti-F monoclonal antibodies (MAbs) that recognize conformation-dependent epitopes. Through heat treatment as a surrogate for H-mediated F triggering, we demonstrate with these MAbs that the morbillivirus F trimer contains a sufficiently high inherent activation energy barrier to maintain the metastable prefusion state even in the absence of H. This notion was further validated by exploring the conformational states of destabilized F mutants and stabilized soluble F variants combined with the use of a membrane fusion inhibitor (3g). Taken together, our findings reveal that the morbillivirus H protein must lower the activation energy barrier of metastable prefusion F for fusion triggering. PMID:23077316

  7. 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.

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

    PubMed

    Schwarzmann, Günter; Breiden, Bernadette; Sandhoff, Konrad

    2015-10-01

    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.

  9. 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

  10. 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

  11. Free energy analysis along the stalk mechanism of membrane fusion.

    PubMed

    Kawamoto, Shuhei; Shinoda, Wataru

    2014-05-07

    The free energy profile of the stalk model of membrane fusion has been calculated using coarse-grained molecular dynamics simulations. The proposed method guides the lipid configuration using a guiding wall potential to make the transition from two apposed membranes to a stalk and a fusion pore. The free energy profile is obtained with a thermodynamic integration scheme using the mean force working on the guiding wall as a response of the system. We applied the method to two apposed flat bilayers composed of dioleoyl phosphatidylethanolamine/dioleoyl phosphatidylcholine expanding over the simulation box under the periodic boundary conditions. The two transition states are identified as pre-stalk and pre-pore states. The free energy barrier for the latter is confirmed to be in good agreement with that estimated by the pulling method. The present method provides a practical way to calculate the free energy profile along the stalk mechanism.

  12. 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

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

    PubMed

    Guardado-Calvo, Pablo; Bignon, Eduardo A; 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

    2016-10-01

    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.

  14. The Gaussian curvature elastic energy of intermediates in membrane fusion.

    PubMed

    Siegel, David P

    2008-12-01

    The Gaussian curvature elastic energy contribution to the energy of membrane fusion intermediates has usually been neglected because the Gaussian curvature elastic modulus, kappa, was unknown. It is now possible to measure kappa for phospholipids that form bicontinuous inverted cubic (Q(II)) phases. Here, it is shown that one can estimate kappa for lipids that do not form Q(II) phases by studying the phase behavior of lipid mixtures. The method is used to estimate kappa for several lipid compositions in excess water. The values of kappa are used to compute the curvature elastic energies of stalks and catenoidal fusion pores according to recent models. The Gaussian curvature elastic contribution is positive and similar in magnitude to the bending energy contribution: it increases the total curvature energy of all the fusion intermediates by 100 units of k(B)T or more. It is important to note that this contribution makes the predicted intermediate energies compatible with observed lipid phase behavior in excess water. An order-of-magnitude fusion rate equation is used to estimate whether the predicted stalk energies are consistent with the observed rates of stalk-mediated processes in pure lipid systems. The current theory predicts a stalk energy that is slightly too large, by approximately 30 k(B)T, to rationalize the observed rates of stalk-mediated processes in phosphatidylethanolamine or N-monomethylated dioleoylphosphatidylethanolamine systems. Despite this discrepancy, the results show that models of fusion intermediate energy are accurate enough to make semiquantitative predictions about how proteins mediate biomembrane fusion. The same rate model shows that for proteins to drive biomembrane fusion at observed rates, they have to perform mediating functions corresponding to a reduction in the energy of a purely lipidic stalk by several tens of k(B)T. By binding particular peptide sequences to the monolayer surface, proteins could lower fusion intermediate

  15. Membrane bending energy and fusion pore kinetics in Ca(2+)-triggered exocytosis.

    PubMed

    Zhang, Zhen; Jackson, Meyer B

    2010-06-02

    A fusion pore composed of lipid is an obligatory kinetic intermediate of membrane fusion, and its formation requires energy to bend membranes into highly curved shapes. The energetics of such deformations in viral fusion is well established, but the role of membrane bending in Ca(2+)-triggered exocytosis remains largely untested. Amperometry recording showed that during exocytosis in chromaffin and PC12 cells, fusion pores formed by smaller vesicles dilated more rapidly than fusion pores formed by larger vesicles. The logarithm of 1/(fusion pore lifetime) varied linearly with vesicle curvature. The vesicle size dependence of fusion pore lifetime quantitatively accounted for the nonexponential fusion pore lifetime distribution. Experimentally manipulating vesicle size failed to alter the size dependence of fusion pore lifetime. Manipulations of membrane spontaneous curvature altered this dependence, and applying the curvature perturbants to the opposite side of the membrane reversed their effects. These effects of curvature perturbants were opposite to those seen in viral fusion. These results indicate that during Ca(2+)-triggered exocytosis membrane bending opposes fusion pore dilation rather than fusion pore formation. Ca(2+)-triggered exocytosis begins with a proteinaceous fusion pore with less stressed membrane, and becomes lipidic as it dilates, bending membrane into a highly curved shape.

  16. An analysis of membrane fusion between mitochondrial double membranes and MITO-Porter, mitochondrial fusogenic vesicles.

    PubMed

    Yamada, Yuma; Fukuda, Yutaka; Harashima, Hideyoshi

    2015-09-01

    To achieve mitochondrial gene therapy, therapeutic molecules need to be transported through the outer and inner membranes of mitochondria into the innermost space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the construction of a MITO-Porter with a high fusogenic activity for the mitochondrial outer membrane for delivering molecules to the mitochondria of human cells. Here, we report on an investigation of a fusogenic lipid composition for the inner membrane, and an analysis of the fusogenic compositions for the outer and inner membranes. A significant relationship was found between fusion activity and the mitochondrial delivery of nucleic acids.

  17. Munc18a Scaffolds SNARE Assembly to Promote Membrane Fusion

    PubMed Central

    Rodkey, Travis L.; Liu, Song; Barry, Meagan

    2008-01-01

    Munc18a is an SM protein required for SNARE-mediated fusion. The molecular details of how Munc18a acts to enhance neurosecretion have remained elusive. Here, we use in vitro fusion assays to characterize how specific interactions between Munc18a and the neuronal SNAREs enhance the rate and extent of fusion. We show that Munc18a interacts directly and functionally with the preassembled t-SNARE complex. Analysis of Munc18a point mutations indicates that Munc18a interacts with helix C of the Syntaxin1a NRD in the t-SNARE complex. Replacement of the t-SNARE SNAP25b with yeast Sec9c had little effect, suggesting that Munc18a has minimal contact with SNAP25b within the t-SNARE complex. A chimeric Syntaxin built of the Syntaxin1a NRD and the H3 domain of yeast Sso1p and paired with Sec9c eliminated stimulation of fusion, suggesting that Munc18a/Syntaxin1a H3 domain contacts are important. Additionally, a Syntaxin1A mutant lacking a flexible linker region that allows NRD movement abolished stimulation of fusion. These experiments suggest that Munc18a binds to the Syntaxin1a NRD and H3 domain within the assembled t-SNARE complex, positioning them for productive VAMP2 binding. In this capacity, Munc18a serves as a platform for trans-SNARE complex assembly that facilitates efficient SNARE-mediated membrane fusion. PMID:18829865

  18. Liposome reconstitution of a minimal protein-mediated membrane fusion machine

    PubMed Central

    Top, Deniz; de Antueno, Roberto; Salsman, Jayme; Corcoran, Jennifer; Mader, Jamie; Hoskin, David; Touhami, Ahmed; Jericho, Manfred H; Duncan, Roy

    2005-01-01

    Biological membrane fusion is dependent on protein catalysts to mediate localized restructuring of lipid bilayers. A central theme in current models of protein-mediated membrane fusion involves the sequential refolding of complex homomeric or heteromeric protein fusion machines. The structural features of a new family of fusion-associated small transmembrane (FAST) proteins appear incompatible with existing models of membrane fusion protein function. While the FAST proteins function to induce efficient cell–cell fusion when expressed in transfected cells, it was unclear whether they function on their own to mediate membrane fusion or are dependent on cellular protein cofactors. Using proteoliposomes containing the purified p14 FAST protein of reptilian reovirus, we now show via liposome–cell and liposome–liposome fusion assays that p14 is both necessary and sufficient for membrane fusion. Stoichiometric and kinetic analyses suggest that the relative efficiency of p14-mediated membrane fusion rivals that of the more complex cellular and viral fusion proteins, making the FAST proteins the simplest known membrane fusion machines. PMID:16079913

  19. 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.

  20. The secretory mechanisms in equine platelets are independent of cytoskeletal polymerization and occur through membrane fusion.

    PubMed

    Brunso, L; Segura, D; Monreal, L; Escolar, G; White, J G; Diaz-Ricart, M

    2010-01-01

    Studies in animal models are useful to understand the basic mechanisms involved in hemostasis and the functional differences among species. Ultrastructural observations led us to predict differences in the activation and secretion mechanisms between equine and human platelets. The potential mechanisms involved have been comparatively explored in the present study. Equine and human platelets were activated with thrombin (0.5 U/ml) and collagen (20 µg/ml), for 90 seconds, and samples processed to evaluate: i) ultrastructural changes, by electron microscopy, ii) actin polymerization and cytoskeletal assembly, by polyacrylamide gel electrophoresis, and iii) specific molecules involved in activation and secretion, by western blot. In activated human platelets, centralization of granules, cytoskeletal assembly and fusion of granules with the open canalicular system were observed. In activated equine platelets, granules fused together forming an organelle chain that fused with the surface membrane and released its content directly outside the platelets. Human platelets responded to activation with actin polymerization and the assembly of other contractile proteins to the cytoskeleton. These events were almost undetectable in equine platelets. When exploring the involvement of the synaptosomal-associated protein-23 (SNAP-23), a known regulator of secretory granule/plasma membrane fusion events, it was present in both human and equine platelets. SNAP-23 was shown to be more activated in equine platelets than human platelets in response to activation, especially with collagen. Thus, there are significant differences in the secretion mechanisms between human and equine platelets. While in human platelets, activation and secretion of granules depend on mechanisms of internal contraction and membrane fusion, in equine platelets the fusion mechanisms seem to be predominant.

  1. 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.

  2. Synaptotagmin-1 C2B domain interacts simultaneously with SNAREs and membranes to promote membrane fusion

    PubMed Central

    Wang, Shen; Li, Yun; Ma, Cong

    2016-01-01

    Synaptotagmin-1 (Syt1) acts as a Ca2+ sensor for neurotransmitter release through its C2 domains. It has been proposed that Syt1 promotes SNARE-dependent fusion mainly through its C2B domain, but the underlying mechanism is poorly understood. In this study, we show that the C2B domain interacts simultaneously with acidic membranes and SNARE complexes via the top Ca2+-binding loops, the side polybasic patch, and the bottom face in response to Ca2+. Disruption of the simultaneous interactions completely abrogates the triggering activity of the C2B domain in liposome fusion. We hypothesize that the simultaneous interactions endow the C2B domain with an ability to deform local membranes, and this membrane-deformation activity might underlie the functional significance of the Syt1 C2B domain in vivo. DOI: http://dx.doi.org/10.7554/eLife.14211.001 PMID:27083046

  3. Henipavirus mediated membrane fusion, virus entry and targeted therapeutics.

    PubMed

    Steffen, Deborah L; Xu, Kai; Nikolov, Dimitar B; Broder, Christopher C

    2012-02-01

    The Paramyxoviridae genus Henipavirus is presently represented by the type species Hendra and Nipah viruses which are both recently emerged zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These enveloped viruses bind and enter host target cells through the coordinated activities of their attachment (G) and class I fusion (F) envelope glycoproteins. The henipavirus G glycoprotein interacts with host cellular B class ephrins, triggering conformational alterations in G that lead to the activation of the F glycoprotein, which facilitates the membrane fusion process. Using the recently published structures of HeV-G and NiV-G and other paramyxovirus glycoproteins, we review the features of the henipavirus envelope glycoproteins that appear essential for mediating the viral fusion process, including receptor binding, G-F interaction, F activation, with an emphasis on G and the mutations that disrupt viral infectivity. Finally, recent candidate therapeutics for henipavirus-mediated disease are summarized in light of their ability to inhibit HeV and NiV entry by targeting their G and F glycoproteins.

  4. Henipavirus Mediated Membrane Fusion, Virus Entry and Targeted Therapeutics

    PubMed Central

    Steffen, Deborah L.; Xu, Kai; Nikolov, Dimitar B.; Broder, Christopher C.

    2012-01-01

    The Paramyxoviridae genus Henipavirus is presently represented by the type species Hendra and Nipah viruses which are both recently emerged zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These enveloped viruses bind and enter host target cells through the coordinated activities of their attachment (G) and class I fusion (F) envelope glycoproteins. The henipavirus G glycoprotein interacts with host cellular B class ephrins, triggering conformational alterations in G that lead to the activation of the F glycoprotein, which facilitates the membrane fusion process. Using the recently published structures of HeV-G and NiV-G and other paramyxovirus glycoproteins, we review the features of the henipavirus envelope glycoproteins that appear essential for mediating the viral fusion process, including receptor binding, G-F interaction, F activation, with an emphasis on G and the mutations that disrupt viral infectivity. Finally, recent candidate therapeutics for henipavirus-mediated disease are summarized in light of their ability to inhibit HeV and NiV entry by targeting their G and F glycoproteins. PMID:22470837

  5. ATP-dependent membrane assembly of F-actin facilitates membrane fusion.

    PubMed

    Jahraus, A; Egeberg, M; Hinner, B; Habermann, A; Sackman, E; Pralle, A; Faulstich, H; Rybin, V; Defacque, H; Griffiths, G

    2001-01-01

    We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.

  6. Visualization and Sequencing of Membrane Remodeling Leading to Influenza Virus Fusion

    PubMed Central

    Gui, Long; Ebner, Jamie L.; Mileant, Alexander; Williams, James A.

    2016-01-01

    ABSTRACT Protein-mediated membrane fusion is an essential step in many fundamental biological events, including enveloped virus infection. The nature of protein and membrane intermediates and the sequence of membrane remodeling during these essential processes remain poorly understood. Here we used cryo-electron tomography (cryo-ET) to image the interplay between influenza virus and vesicles with a range of lipid compositions. By following the population kinetics of membrane fusion intermediates imaged by cryo-ET, we found that membrane remodeling commenced with the hemagglutinin fusion protein spikes grappling onto the target membrane, followed by localized target membrane dimpling as local clusters of hemagglutinin started to undergo conformational refolding. The local dimples then transitioned to extended, tightly apposed contact zones where the two proximal membrane leaflets were in most cases indistinguishable from each other, suggesting significant dehydration and possible intermingling of the lipid head groups. Increasing the content of fusion-enhancing cholesterol or bis-monoacylglycerophosphate in the target membrane led to an increase in extended contact zone formation. Interestingly, hemifused intermediates were found to be extremely rare in the influenza virus fusion system studied here, most likely reflecting the instability of this state and its rapid conversion to postfusion complexes, which increased in population over time. By tracking the populations of fusion complexes over time, the architecture and sequence of membrane reorganization leading to efficient enveloped virus fusion were thus resolved. IMPORTANCE Enveloped viruses employ specialized surface proteins to mediate fusion of cellular and viral membranes that results in the formation of pores through which the viral genetic material is delivered to the cell. For influenza virus, the trimeric hemagglutinin (HA) glycoprotein spike mediates host cell attachment and membrane fusion. While

  7. Divergent pathways lead to ESCRT-III-catalyzed membrane fission.

    PubMed

    Peel, Suman; Macheboeuf, Pauline; Martinelli, Nicolas; Weissenhorn, Winfried

    2011-04-01

    Endosomal sorting complexes required for transport (ESCRT) have been implicated in topologically similar but diverse cellular and pathological processes including multivesicular body (MVB) biogenesis, cytokinesis and enveloped virus budding. Although receptor sorting at the endosomal membrane producing MVBs employs the regulated assembly of ESCRT-0 followed by ESCRT-I, -II, -III and the vacuolar protein sorting (VPS)4 complex, other ESCRT-catalyzed processes require only a subset of complexes which commonly includes ESCRT-III and VPS4. Recent progress has shed light on the pathway of ESCRT assembly and highlights the separation of tasks of different ESCRT complexes and associated partners. The emerging picture suggests that among all ESCRT-catalyzed processes, divergent pathways lead to ESCRT-III assembly within the neck of a budding structure catalyzing membrane fission.

  8. 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

  9. 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.

  10. Base of the Measles Virus Fusion Trimer Head Receives the Signal That Triggers Membrane Fusion*

    PubMed Central

    Apte-Sengupta, Swapna; Negi, Surendra; Leonard, Vincent H. J.; Oezguen, Numan; Navaratnarajah, Chanakha K.; Braun, Werner; Cattaneo, Roberto

    2012-01-01

    The measles virus (MV) fusion (F) protein trimer executes membrane fusion after receiving a signal elicited by receptor binding to the hemagglutinin (H) tetramer. Where and how this signal is received is understood neither for MV nor for other paramyxoviruses. Because only the prefusion structure of the parainfluenza virus 5 (PIV5) F-trimer is available, to study signal receipt by the MV F-trimer, we generated and energy-refined a homology model. We used two approaches to predict surface residues of the model interacting with other proteins. Both approaches measured interface propensity values for patches of residues. The second approach identified, in addition, individual residues based on the conservation of physical chemical properties among F-proteins. Altogether, about 50 candidate interactive residues were identified. Through iterative cycles of mutagenesis and functional analysis, we characterized six residues that are required specifically for signal transmission; their mutation interferes with fusion, although still allowing efficient F-protein processing and cell surface transport. One residue is located adjacent to the fusion peptide, four line a cavity in the base of the F-trimer head, while the sixth residue is located near this cavity. Hydrophobic interactions in the cavity sustain the fusion process and contacts with H. The cavity is flanked by two different subunits of the F-trimer. Tetrameric H-stalks may be lodged in apposed cavities of two F-trimers. Because these insights are based on a PIV5 homology model, the signal receipt mechanism may be conserved among paramyxoviruses. PMID:22859308

  11. 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

  12. In vitro fusion of lung lamellar bodies and plasma membrane is augmented by lung synexin.

    PubMed

    Chander, A; Wu, R D

    1991-11-05

    Lamellar bodies of lung epithelial type II cells undergo fusion with plasma membrane prior to exocytosis of surfactant into the alveolar lumen. Since synexin from adrenal glands promotes aggregation and fusion of chromaffin granules, we purified synexin-like proteins from bovine lung cytosolic fraction, and evaluated their effect on the fusion of isolated lamellar bodies and plasma membrane fractions. Synexin activity, which co-purified with an approx. 47 kDa protein (pI 6.8), was assessed by following calcium-dependent aggregation of liposomes prepared from a mixture of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1, mol/mol). Lung synexin caused aggregation of liposomes approximating lung surfactant lipid-like composition, isolated lamellar bodies, or isolated plasma membrane fraction. Lung synexin promoted fusion only in the presence of calcium. It augmented fusion between lamellar bodies and plasma membranes, lamellar bodies and liposomes, or between two populations of liposomes. However, selectivity with regard to synexin-mediated fusion was observed as synexin did not promote fusion between plasma membrane and liposomes, or between liposomes of surfactant lipid-like composition and other liposomes. These observations support a role for lung synexin in membrane fusion between the plasma membrane and lamellar bodies during exocytosis of lung surfactant, and suggest that such fusion is dependent on composition of interacting membranes.

  13. Myomaker is a membrane activator of myoblast fusion and muscle formation.

    PubMed

    Millay, Douglas P; O'Rourke, Jason R; Sutherland, Lillian B; Bezprozvannaya, Svetlana; Shelton, John M; Bassel-Duby, Rhonda; Olson, Eric N

    2013-07-18

    Fusion of myoblasts is essential for the formation of multi-nucleated muscle fibres. However, the identity of muscle-specific proteins that directly govern this fusion process in mammals has remained elusive. Here we identify a muscle-specific membrane protein, named myomaker, that controls myoblast fusion. Myomaker is expressed on the cell surface of myoblasts during fusion and is downregulated thereafter. Overexpression of myomaker in myoblasts markedly enhances fusion, and genetic disruption of myomaker in mice causes perinatal death due to an absence of multi-nucleated muscle fibres. Remarkably, forced expression of myomaker in fibroblasts promotes fusion with myoblasts, demonstrating the direct participation of this protein in the fusion process. Pharmacological perturbation of the actin cytoskeleton abolishes the activity of myomaker, consistent with previous studies implicating actin dynamics in myoblast fusion. These findings reveal a long-sought myogenic fusion protein that controls mammalian myoblast fusion and provide new insights into the molecular underpinnings of muscle formation.

  14. 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.

  15. 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.

  16. SARS-CoV fusion peptides induce membrane surface ordering and curvature.

    PubMed

    Basso, Luis G M; Vicente, Eduardo F; Crusca, Edson; Cilli, Eduardo M; Costa-Filho, Antonio J

    2016-11-28

    Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

  17. SARS-CoV fusion peptides induce membrane surface ordering and curvature

    PubMed Central

    Basso, Luis G. M.; Vicente, Eduardo F.; Crusca Jr., Edson; Cilli, Eduardo M.; Costa-Filho, Antonio J.

    2016-01-01

    Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed. PMID:27892522

  18. 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

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

    PubMed

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

    2016-07-19

    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.

  20. 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.

  1. 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

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

    PubMed

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

    2015-10-13

    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.

  3. 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

  4. 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.

  5. Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface

    PubMed Central

    1979-01-01

    The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze- fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed. PMID:582596

  6. 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

    2015-06-01

    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.

  7. 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.

  8. How and why intralumenal membrane fragments form during vacuolar lysosome fusion

    PubMed Central

    Mattie, Sevan; McNally, Erin K.; Karim, Mahmoud A.; Vali, Hojatollah; Brett, Christopher L.

    2017-01-01

    Lysosomal membrane fusion mediates the last step of the autophagy and endocytosis pathways and supports organelle remodeling and biogenesis. Because fusogenic proteins and lipids concentrate in a ring at the vertex between apposing organelle membranes, the encircled area of membrane can be severed and internalized within the lumen as a fragment upon lipid bilayer fusion. How or why this intralumenal fragment forms during fusion, however, is not entirely clear. To better understand this process, we studied fragment formation during homotypic vacuolar lysosome membrane fusion in Saccharomyces cerevisiae. Using cell-free fusion assays and light microscopy, we find that GTPase activation and trans-SNARE complex zippering have opposing effects on fragment formation and verify that this affects the morphology of the fusion product and regulates transporter protein degradation. We show that fragment formwation is limited by stalk expansion, a key intermediate of the lipid bilayer fusion reaction. Using electron microscopy, we present images of hemifusion diaphragms that form as stalks expand and propose a model describing how the fusion machinery regulates fragment formation during lysosome fusion to control morphology and protein lifetimes. PMID:27881666

  9. Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles

    PubMed Central

    Espósito, Danillo L. A.; Nguyen, Jennifer B.; DeWitt, David C.; Rhoades, Elizabeth

    2015-01-01

    Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7–20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process. PMID:25740960

  10. 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

  11. Mitochondrial DNA mutations provoke dominant inhibition of mitochondrial inner membrane fusion.

    PubMed

    Sauvanet, Cécile; Duvezin-Caubet, Stéphane; Salin, Bénédicte; David, Claudine; Massoni-Laporte, Aurélie; di Rago, Jean-Paul; Rojo, Manuel

    2012-01-01

    Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of

  12. Dual Split Protein (DSP) Assay to Monitor Cell-Cell Membrane Fusion.

    PubMed

    Nakane, Shuhei; Matsuda, Zene

    2015-01-01

    Fusion between viral and cellular membranes is the essential first step in infection of enveloped viruses. This step is mediated by viral envelope glycoproteins (Env) that recognize cellular receptors. The membrane fusion between the effector cells expressing viral Env and the target cells expressing its receptors can be monitored by several methods. We have recently developed a pair of chimeric reporter protein composed of split Renilla luciferase (RL) and split GFP. We named this reporter dual split protein (DSP), since it recovers both RL and GFP activities upon self reassociation. By using DSP, pore formation and content mixing between the effector and target cells can be monitored upon the recovery of RL and GFP activities after the membrane fusion. This quick assay provides quantitative as well as spatial information about membrane fusion mediated by viral Env.

  13. Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers.

    PubMed

    Paxman, Jason; Hunt, Brady; Hallan, David; Zarbock, Samuel R; Woodbury, Dixon J

    2017-01-10

    Although the effects of ethanol on protein receptors and lipid membranes have been studied extensively, ethanol's effect on vesicles fusing to lipid bilayers is not known. To determine the effect of alcohols on fusion rates, we utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a planar lipid bilayer (BLM). The addition of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited fusion on the trans side. Other short-chain alcohols followed a similar pattern. In general, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory (cis) effect, with a decrease of 29% in fusion rates at the legal driving limit of 0.08% (w/v) ethanol (IC50 = 0.2% v/v, 34 mM). Similar inhibitory effects were observed with methanol, propanol, and butanol, with ethanol being the most potent. Significant variability was observed with different alcohols when applied to the cis side. Ethanol and propanol enhanced fusion, butanol also enhanced fusion but was less potent, and low doses of methanol mildly inhibited fusion. The inhibition by trans addition of alcohols implies that they alter the planar membrane structure and thereby increase the activation energy required for fusion, likely through an increase in membrane fluidity. The cis data are likely a combination of the above effect and a proportionally greater lowering of the vesicle lysis tension and hydration repulsive pressure that combine to enhance fusion. Alternate hypotheses are also discussed. The inhibitory effect of ethanol on liposome-membrane fusion is large enough to provide a possible biophysical explanation of compromised neuronal behavior.

  14. 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.

  15. 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).

  16. 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.

  17. Measuring the Strength of Interaction between the Ebola Fusion Peptide and Lipid Rafts: Implications for Membrane Fusion and Virus Infection

    PubMed Central

    Freitas, Mônica S.; Follmer, Cristian; Costa, Lilian T.; Vilani, Cecília; Bianconi, M. Lucia; Achete, Carlos Alberto; Silva, Jerson L.

    2011-01-01

    The Ebola fusion peptide (EBO16) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO16 and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO16 to induce lipid mixing. On the other hand, EBO16 was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO16. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO16 and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases. PMID:21249196

  18. LC3 and GATE-16 N termini mediate membrane fusion processes required for autophagosome biogenesis.

    PubMed

    Weidberg, Hilla; Shpilka, Tomer; Shvets, Elena; Abada, Adi; Shimron, Frida; Elazar, Zvulun

    2011-04-19

    Autophagy is a unique membrane trafficking pathway describing the formation and targeting of double membrane autophagosomes to the vacuole/lysosome. The biogenesis of autophagosomes and their delivery to the vacuole/lysosome depend on multiple membrane fusion events. Using a cell-free system, we have investigated the ability of LC3 and GATE-16, two mammalian Atg8 orthologs, to mediate membrane fusion. We found that both proteins promote tethering and membrane fusion, mediated by the proteins' N-terminal α helices. We further show that short, 10 amino acid long synthetic peptides derived from the N terminus of LC3 or GATE-16 are sufficient to promote membrane fusion. Our data indicate that the fusion activity of LC3 is mediated by positively charged amino acids, whereas the activity of GATE-16 is mediated by hydrophobic interactions. Finally, we demonstrate that LC3 and GATE-16 N termini in general and specific residues needed for the fusion activity are essential for the proteins role in autophagosome biogenesis.

  19. Transforaminal lumbar interbody fusion versus instrumented posterolateral fusion in Grade I/II spondylolisthesis

    PubMed Central

    Pooswamy, Shanmugasundaram; Muralidharagopalan, Niranjanan Raghavn; Subbaiah, Sivasubramaniam

    2017-01-01

    Background: Spondylolisthesis refers to slippage of one vertebra over the other, which may be caused by a variety of reasons such as degenerative, trauma, and isthmic. Surgical management forms the mainstay of treatment to prevent further slip and worsening. However, there is no consensus regarding the best surgical option to treat these patients. This study compares TLIF and instrumented PLF in patients with Grade I and II spondylolisthesis and analysis the outcome with respect to functional outcome, pain, fusion rate, adequacy of medial facetectomy for decompression, and complications. Materials and Methods: Forty patients operated for spondylolisthesis by instrumented posterolateral or transforaminal fusion between January 1, 2010, and June 30, 2012 were included in this retrospective study. They were followed up for 3 years. Twenty one cases were of instrumented posterolateral fusion (PLF) and 19 cases were of transforaminal lumbar interbody fusion (TLIF). The patients were asked to fill up the Oswestry disability index (ODI), Dallas Pain Questionnaire (DPQ), and low back pain rating scale (LBPRS) preoperatively, at 1-month postoperatively, and at 6, 12, 24, and 36 months postoperatively. Radiological parameters were assessed using radiographs. Results: No significant differences were found in DPQ, LBPRS, or ODI scores preoperative, 1-month postoperative, and at 6, 12, 24 and 36 months followup. No significant difference was found between the two groups in blood loss. The only significant difference between the two groups was in the operative time, in which the instrumented PLF group had a mean of 50 min lesser than the TLIF group (P = 0.02). Conclusions: TLIF and instrumented PLF are equally efficacious options in the treatment of Grade I and II spondylolisthesis, except lytic type.

  20. Membrane penetration of Sendai virus glycoproteins during the early stages of fusion with liposomes as determined by hydrophobic photoaffinity labeling

    SciTech Connect

    Novick, S.L.; Hoekstra, D.

    1988-10-01

    The hydrophobic photoaffinity label 3-(trifluoromethyl)-3-(m-(/sup 125/I)iodophenyl)diazirine was used to label Sendai virus proteins during fusion with cardiolipin and phosphatidylserine liposomes. Preferential labeling of the viral fusion protein during the initial stages of fusion demonstrated that this protein interacts with the hydrophobic core of the target membrane as an initiating event of virus-liposome fusion. Labeling showed time, temperature, and pH dependence consistent with earlier fluorescent measurements of fusion kinetics. The present method provides conclusive evidence supporting the hypothesis that hydrophobic interaction of the fusion protein with the target bilayer is an initial event in the fusion mechanism of viral membranes.

  1. High cholesterol obviates a prolonged hemifusion intermediate in fast SNARE-mediated membrane fusion.

    PubMed

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

    2015-07-21

    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.

  2. The fusion of membranes and vesicles: pathway and energy barriers from dissipative particle dynamics.

    PubMed

    Grafmüller, Andrea; Shillcock, Julian; Lipowsky, Reinhard

    2009-04-08

    The fusion of lipid bilayers is studied with dissipative particle dynamics simulations. First, to achieve control over membrane properties, the effects of individual simulation parameters are studied and optimized. Then, a large number of fusion events for a vesicle and a planar bilayer are simulated using the optimized parameter set. In the observed fusion pathway, configurations of individual lipids play an important role. Fusion starts with individual lipids assuming a splayed tail configuration with one tail inserted in each membrane. To determine the corresponding energy barrier, we measure the average work for interbilayer flips of a lipid tail, i.e., the average work to displace one lipid tail from one bilayer to the other. This energy barrier is found to depend strongly on a certain dissipative particle dynamics parameter, and, thus, can be adjusted in the simulations. Overall, three subprocesses have been identified in the fusion pathway. Their energy barriers are estimated to lie in the range 8-15 k(B)T. The fusion probability is found to possess a maximum at intermediate tension values. As one decreases the tension, the fusion probability seems to vanish before the tensionless membrane state is attained. This would imply that the tension has to exceed a certain threshold value to induce fusion.

  3. 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.

  4. 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.

  5. Folded monomers and hexamers of the ectodomain of the HIV gp41 membrane fusion protein: potential roles in fusion and synergy between the fusion peptide, hairpin, and membrane-proximal external region.

    PubMed

    Banerjee, Koyeli; Weliky, David P

    2014-11-25

    HIV is an enveloped virus and fusion between the HIV and host cell membranes is catalyzed by the ectodomain of the HIV gp41 membrane protein. Both the N-terminal fusion peptide (FP) and C-terminal membrane-proximal external region (MPER) are critical for fusion and are postulated to bind to the host cell and HIV membranes, respectively. Prior to fusion, the gp41 on the virion is a trimer in noncovalent complex with larger gp120 subunits. The gp120 bind host cell receptors and move away or dissociate from gp41 which subsequently catalyzes fusion. In the present work, large gp41 ectodomain constructs were produced and biophysically and structurally characterized. One significant finding is observation of synergy between the FP, hairpin, and MPER in vesicle fusion. The ectodomain-induced fusion can be very efficient with only ∼15 gp41 per vesicle, which is comparable to the number of gp41 on a virion. Conditions are found with predominant monomer or hexamer but not trimer and these may be oligomeric states during fusion. Monomer gp41 ectodomain is hyperthermostable and has helical hairpin structure. A new HIV fusion model is presented where (1) hemifusion is catalyzed by folding of gp41 ectodomain monomers into hairpins and (2) subsequent fusion steps are catalyzed by assembly into a hexamer with FPs in an antiparallel β sheet. There is also significant interest in the gp41 MPER because it is the epitope of several broadly neutralizing antibodies. Two of these antibodies bind our gp41 ectodomain constructs and support investigation of the gp41 ectodomain as an immunogen in HIV vaccine development.

  6. Evidence for thylakoid membrane fusion during zygote formation in Chlamydomonas reinhardtii

    PubMed Central

    1991-01-01

    To understand whether fusions of thylakoid membranes from the parental chloroplasts occurred during zygote formation in Chlamydomonas reinhardtii, we performed an ultrastructural analysis of the zygotes produced by crossing mutants lacking photosystem I or II protein complexes, in the absence of de novo chloroplast protein synthesis. Thylakoid membranes from each parent could be distinguished on thin sections due to their organization in "supergrana" in mutants lacking photosystem I centers, by freeze-fracturing due to the absence of most of the exoplasmic-face (EF) particles in mutants lacking photosystem II centers, by immunocytochemistry using antibodies directed against photosystem II subunits. We demonstrate that a fusion of the thylakoid membranes occurred during zygote formation approximately 15 h after mating. These fusions allowed a lateral redistribution of the thylakoid membrane proteins. These observations provide the structural basis for the restoration of photosynthetic electron flow in the mature zygote that we observed in fluorescence induction experiments. PMID:1874788

  7. 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.

  8. Rational design of a fusion partner for membrane protein expression in E. coli

    PubMed Central

    Luo, Jianying; Choulet, Julie; Samuelson, James C

    2009-01-01

    We have designed a novel protein fusion partner (P8CBD) to utilize the co-translational SRP pathway in order to target heterologous proteins to the E. coli inner membrane. SRP-dependence was demonstrated by analyzing the membrane translocation of P8CBD-PhoA fusion proteins in wt and SRP-ffh77 mutant cells. We also demonstrate that the P8CBD N-terminal fusion partner promotes over-expression of a Thermotoga maritima polytopic membrane protein by replacement of the native signal anchor sequence. Furthermore, the yeast mitochondrial inner membrane protein Oxa1p was expressed as a P8CBD fusion and shown to function within the E. coli inner membrane. In this example, the mitochondrial targeting peptide was replaced by P8CBD. Several practical features were incorporated into the P8CBD expression system to aid in protein detection, purification, and optional in vitro processing by enterokinase. The basis of membrane protein over-expression toxicity is discussed and solutions to this problem are presented. We anticipate that this optimized expression system will aid in the isolation and study of various recombinant forms of membrane-associated protein. PMID:19530231

  9. Membrane interactions of fusogenic coiled-coil peptides: implications for lipopeptide mediated vesicle fusion.

    PubMed

    Rabe, Martin; Schwieger, Christian; Zope, Harshal R; Versluis, Frank; Kros, Alexander

    2014-07-08

    Fusion of lipid membranes is an important natural process for the intra- and intercellular exchange of molecules. However, little is known about the actual fusion mechanism at the molecular level. In this study we examine a system that models the key features of this process. For the molecular recognition between opposing membranes two membrane anchored heterodimer coiled-coil forming peptides called 'E' (EIAALEK)3 and 'K' (KIAALKE)3 were used. Lipid monolayers and IR reflection absorption spectroscopy (IRRAS) revealed the interactions of the peptides 'E', 'K', and their parallel coiled-coil complex 'E/K' with the phospholipid membranes and thereby mimicked the pre- and postfusion states, respectively. The peptides adopted α-helical structures and were incorporated into the monolayers with parallel orientation. The strength of binding to the monolayer differed for the peptides and tethering them to the membrane increased the interactions even further. Remarkably, these interactions played a role even in the postfusion state. These findings shed light on important mechanistic details of the membrane fusion process in this model system. Furthermore, their implications will help to improve the rational design of new artificial membrane fusion systems, which have a wide range of potential applications in supramolecular chemistry and biomedicine.

  10. Low energy cost for optimal speed and control of membrane fusion.

    PubMed

    François-Martin, Claire; Rothman, James E; Pincet, Frederic

    2017-02-07

    Membrane fusion is the cell's delivery process, enabling its many compartments to receive cargo and machinery for cell growth and intercellular communication. The overall activation energy of the process must be large enough to prevent frequent and nonspecific spontaneous fusion events, yet must be low enough to allow it to be overcome upon demand by specific fusion proteins [such as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs)]. Remarkably, to the best of our knowledge, the activation energy for spontaneous bilayer fusion has never been measured. Multiple models have been developed and refined to estimate the overall activation energy and its component parts, and they span a very broad range from 20 kBT to 150 kBT, depending on the assumptions. In this study, using a bulk lipid-mixing assay at various temperatures, we report that the activation energy of complete membrane fusion is at the lowest range of these theoretical values. Typical lipid vesicles were found to slowly and spontaneously fully fuse with activation energies of ∼30 kBT Our data demonstrate that the merging of membranes is not nearly as energy consuming as anticipated by many models and is ideally positioned to minimize spontaneous fusion while enabling rapid, SNARE-dependent fusion upon demand.

  11. Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18.

    PubMed

    Zick, Michael; Orr, Amy; Schwartz, Matthew L; Merz, Alexey J; Wickner, William T

    2015-05-05

    Sec17 [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17(L291A,L292A) did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17(F21S,M22S), with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17(F21S,M22S), interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.

  12. Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18

    PubMed Central

    Zick, Michael; Orr, Amy; Schwartz, Matthew L.; Merz, Alexey J.; Wickner, William T.

    2015-01-01

    Sec17 [soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17F21S,M22S, with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17F21S,M22S, interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion. PMID:25902545

  13. 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.

  14. 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

  15. Effect of physical constraints on the mechanisms of membrane fusion: bolaform lipid vesicles as model systems.

    PubMed Central

    Relini, A; Cassinadri, D; Fan, Q; Gulik, A; Mirghani, Z; De Rosa, M; Gliozzi, A

    1996-01-01

    Bolaform lipid vesicles were used to study the effect of physical constraints on membrane fusion. In these vesicles the membrane is organized in a single monolayer, because of the presence of covalent bonds in its middle plane. Therefore, the formation of fusion intermediates is subject to higher energy barriers and greater geometrical constraints than is usual in bilayer membranes. Bolaform lipids were extracted from the thermophilic archaeon Sulfolobus solfataricus. These lipids can be divided into two classes, the monosubstituted molecules, in which one of the polar heads is glycerol, and the bisubstituted molecules, endowed with two complex polar heads. The fusion process in vesicles composed of different mixtures of monosubstituted/bisubstituted molecules was studied by means of fluorescence techniques. Ca2+ or poly(ethylene glycol) was employed as a fusogenic agent. We found that fusion of such constrained membranes is still possible, provided that molecules able to mediate a structural rearrangement of the membrane are present. This condition is fulfilled by monosubstituted molecules, which are able to partition the glycerol headgroup in the apolar moiety. In addition, the presence of traces (approximately 5%) of the monopolar compound diphytanylglycerol is an important factor for fusion to occur. On the contrary, vesicles formed by bisubstituted molecules are unable to fuse, irrespective of the fusogen employed. Images FIGURE 2 PMID:8889155

  16. pH-dependent vesicle fusion induced by the ectodomain of the human immunodeficiency virus membrane fusion protein gp41: Two kinetically distinct processes and fully-membrane-associated gp41 with predominant β sheet fusion peptide conformation.

    PubMed

    Ratnayake, Punsisi U; Sackett, Kelly; Nethercott, Matthew J; Weliky, David P

    2015-01-01

    The gp41 protein of the Human Immunodeficiency Virus (HIV) catalyzes fusion between HIV and host cell membranes. The ~180-residue ectodomain of gp41 is outside the virion and is the most important gp41 region for membrane fusion. The ectodomain consists of an apolar fusion peptide (FP) region hypothesized to bind to the host cell membrane followed by N-heptad repeat (NHR), loop, and C-heptad repeat (CHR) regions. The present study focuses on the large gp41 ectodomain constructs "Hairpin" (HP) containing NHR+loop+CHR and "FP-Hairpin" (FP-HP) containing FP+NHR+loop+CHR. Both proteins induce rapid and extensive fusion of anionic vesicles at pH4 where the protein is positively-charged but do not induce fusion at pH7 where the protein is negatively charged. This observation, along with lack of fusion of neutral vesicles at either pH supports the significance of attractive protein/membrane electrostatics in fusion. There are two kinetically distinct fusion processes at pH4: (1) a faster ~100 ms⁻¹ process with rate strongly positively correlated with vesicle charge; and (2) a slower ~5 ms⁻¹ process with extent strongly inversely correlated with this charge. The slower process may be more physiologically relevant because HIV/host cell fusion occurs at physiologic pH with gp41 restricted to the narrow region between the two membranes. Previous solid-state NMR (SSNMR) of membrane-associated FP-HP has supported protein oligomers with FP's in an intermolecular antiparallel sheet. There was an additional population of molecules with α helical FPs and the samples likely contained a mixture of membrane-bound and -unbound proteins. For the present study, samples were prepared with fully membrane-bound FP-HP and subsequent SSNMR showed dominant β FP conformation at both low and neutral pH. SSNMR also showed close contact of the FP with the lipid headgroups at both low and neutral pH whereas the NHR+CHR regions had contact at low pH and were more distant at neutral p

  17. 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.

  18. Specific interaction of IM30/Vipp1 with cyanobacterial and chloroplast membranes results in membrane remodeling and eventually in membrane fusion.

    PubMed

    Heidrich, Jennifer; Thurotte, Adrien; Schneider, Dirk

    2017-04-01

    The photosynthetic light reaction takes place within the thylakoid membrane system in cyanobacteria and chloroplasts. Besides its global importance, the biogenesis, maintenance and dynamics of this membrane system are still a mystery. In the last two decades, strong evidence supported the idea that these processes involve IM30, the inner membrane-associated protein of 30kDa, a protein also known as the vesicle-inducing protein in plastids 1 (Vipp1). Even though we just only begin to understand the precise physiological function of this protein, it is clear that interaction of IM30 with membranes is crucial for biogenesis of thylakoid membranes. Here we summarize and discuss forces guiding IM30-membrane interactions, as the membrane properties as well as the oligomeric state of IM30 appear to affect proper interaction of IM30 with membrane surfaces. Interaction of IM30 with membranes results in an altered membrane structure and can finally trigger fusion of adjacent membranes, when Mg(2+) is present. Based on recent results, we finally present a model summarizing individual steps involved in IM30-mediated membrane fusion. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider.

  19. A dynamin homolog promotes the transition from hemifusion to content mixing in intracellular membrane fusion

    PubMed Central

    Kulkarni, Aditya; Alpadi, Kannan; Sirupangi, Tirupataiah; Peters, Christopher

    2014-01-01

    The convergence of the antagonistic reactions of membrane fusion and fission at the hemifusion/hemifission intermediate has generated a captivating enigma of whether SNAREs and dynamin have unusual counter-functions in fission and fusion respectively. SNARE-mediated fusion and dynamin-driven fission are fundamental membrane flux reactions known to occur during ubiquitous cellular communication events such as exocytosis, endocytosis and vesicle transport. Here we demonstrate the influence of the dynamin homolog Vps1 on lipid mixing and content mixing properties of yeast vacuoles, and on the incorporation of SNAREs into fusogenic complexes. We propose a novel concept that Vps1, through its oligomerization and SNARE domain binding, promotes the hemifusion-content mixing transition in yeast vacuole fusion by increasing the number of trans-SNAREs. PMID:24471450

  20. 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.

  1. Fusion peptide from influenza hemagglutinin increases membrane surface order: an electron-spin resonance study.

    PubMed

    Ge, Mingtao; Freed, Jack H

    2009-06-17

    A spin-labeling study of interactions of a fusion peptide from the hemagglutinin of the influenza virus, wt20, and a fusion-inactive mutant DeltaG1 with dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoyl-phosphatdylcholine bilayers was performed. We found that upon binding of wt20, the ordering of headgroups and the ordering of acyl chains near the headgroup increased significantly, in a manner consistent with a cooperative phenomenon. However, changes in the order at the end of the acyl chains were negligible. The ordering effect of wt20 on the headgroup was much stronger at pH 5 than at pH 7. No effect of DeltaG1 binding on the order of bilayers was evident. We also found that 1-palmitoyl-2-hydroxyl phosphatidylcholine, a membrane-fusion inhibitor, decreased the ordering of DMPC headgroups, whereas arachidonic acid, a membrane-fusion promoter, increased the ordering of DMPC headgroups. These results suggest that increases in headgroup ordering may be important for membrane fusion. We propose that upon binding of wt20, which is known to affect only the outer leaflet of the bilayer, this outer leaflet becomes more ordered, and thus more solid-like. Then the coupling between the hardened outer leaflet and the softer inner leaflet generates bending stresses in the bilayer, which tend to increase the negative curvature of the bilayer. We suggest that the increased ordering in the headgroup region enhances dipolar interactions and lowers electrostatic energy, which may provide an energy source for membrane fusion. Possible roles of bending stresses in promoting membrane fusion are discussed.

  2. Atomic force microscopy: Unraveling the fundamental principles governing secretion and membrane fusion in cells.

    PubMed

    Jena, Bhanu P

    2009-07-01

    The story of cell secretion and membrane fusion is as old as life itself. Without these fundamental cellular processes known to occur in yeast to humans, life would cease to exist. In the last 15 years, primarily using the atomic force microscope, a detailed understanding of the molecular process and of the molecular machinery and mechanism of secretion and membrane fusion in cells has come to light. This has led to a paradigm shift in our understanding of the underlying mechanism of cell secretion. The journey leading to the discovery of a new cellular structure the 'porosome',-the universal secretory machinery in cells, and the contributions of the AFM in our understanding of the general molecular machinery and mechanism of cell secretion and membrane fusion, is briefly discussed in this article.

  3. SNARE-mediated membrane fusion trajectories derived from force-clamp experiments.

    PubMed

    Oelkers, Marieelen; Witt, Hannes; Halder, Partho; Jahn, Reinhard; Janshoff, Andreas

    2016-11-15

    Fusion of lipid bilayers is usually prevented by large energy barriers arising from removal of the hydration shell, formation of highly curved structures, and, eventually, fusion pore widening. Here, we measured the force-dependent lifetime of fusion intermediates using membrane-coated silica spheres attached to cantilevers of an atomic-force microscope. Analysis of time traces obtained from force-clamp experiments allowed us to unequivocally assign steps in deflection of the cantilever to membrane states during the SNARE-mediated fusion with solid-supported lipid bilayers. Force-dependent lifetime distributions of the various intermediate fusion states allowed us to propose the likelihood of different fusion pathways and to assess the main free energy barrier, which was found to be related to passing of the hydration barrier and splaying of lipids to eventually enter either the fully fused state or a long-lived hemifusion intermediate. The results were compared with SNARE mutants that arrest adjacent bilayers in the docked state and membranes in the absence of SNAREs but presence of PEG or calcium. Only with the WT SNARE construct was appreciable merging of both bilayers observed.

  4. 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

  5. Fusion of artificial membranes with mammalian spermatozoa. Specific involvement of the equatorial segment after acrosome reaction.

    PubMed

    Arts, E G; Kuiken, J; Jager, S; Hoekstra, D

    1993-11-01

    The fusogenic properties of bovine and human spermatozoa membranes were investigated, using phospholipid bilayers (liposomes) as target membranes. Fusion was monitored by following lipid mixing, as revealed by an assay based on resonance-energy transfer. In addition, fusion was visualized by fluorescence microscopy, using fluorescent lipid vesicles. Cryopreserved bovine sperm fused with liposomes before induction of the acrosome reaction, fluorescence being located in essentially all spermatozoa membrane domains. Fresh bovine and human spermatozoa fused with liposomes only after the induction of the acrosome reaction, as triggered by calcium ionophore A23187 or zonae pellucidae (proteins), while the fluorescence distribution was mainly restricted to the equatorial segment (ES). However, with spermatozoa that had undergone a freeze/thawing cycle, domains other than ES also became labeled. Hence, the redistribution of the lipid probes over the entire membrane occurring during lipid mixing with cryopreserved bovine sperm is probably related to membrane perturbations caused by long-term cryopreservation. Fusion with liposomes was governed by spermatozoa factors and required the presence of acidic phospholipids like cardiolipin and phosphatidylserine in the liposomal bilayer. Incorporation of the zwitterionic lipid phosphatidylcholine in the vesicles inhibited the fusion reaction. Fusion was pH dependent. The results indicate that the ES is the primary domain of spermatozoa membranes that harbours the fusogenic capacity of sperm. Liposomes appear a valuable tool in further characterizing the properties of this domain, which has been claimed [Yanagimachi, R. (1988) in The physiology of reproduction (Knobil, E. & Neill, J., eds) pp. 135-185, Raven Press, New York] to represent the putative, initial fusion site for the oocyte.

  6. Appoptosin interacts with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

    PubMed

    Zhang, Cuilin; Shi, Zhun; Zhang, Lingzhi; Zhou, Zehua; Zheng, Xiaoyuan; Liu, Guiying; Bu, Guojun; Fraser, Paul E; Xu, Huaxi; Zhang, Yun-Wu

    2016-03-01

    Mitochondrial morphology is regulated by fusion and fission machinery. Impaired mitochondria dynamics cause various diseases, including Alzheimer's disease. Appoptosin (encoded by SLC25A38) is a mitochondrial carrier protein that is located in the mitochondrial inner membrane. Appoptosin overexpression causes overproduction of reactive oxygen species (ROS) and caspase-dependent apoptosis, whereas appoptosin downregulation abolishes β-amyloid-induced mitochondrial fragmentation and neuronal death during Alzheimer's disease. Herein, we found that overexpression of appoptosin resulted in mitochondrial fragmentation in a manner independent of its carrier function, ROS production or caspase activation. Although appoptosin did not affect levels of mitochondrial outer-membrane fusion (MFN1 and MFN2), inner-membrane fusion (OPA1) and fission [DRP1 (also known as DNM1L) and FIS1] proteins, appoptosin interacted with MFN1 and MFN2, as well as with the mitochondrial ubiquitin ligase MITOL (also known as MARCH5) but not OPA1, FIS1 or DRP1. Appoptosin overexpression impaired the interaction between MFN1 and MFN2, and mitochondrial fusion. By contrast, co-expression of MFN1, MITOL and a dominant-negative form of DRP1, DRP1(K38A), partially rescued appoptosin-induced mitochondrial fragmentation and apoptosis, whereas co-expression of FIS1 aggravated appoptosin-induced apoptosis. Together, our results demonstrate that appoptosin can interact with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

  7. In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles.

    PubMed Central

    Arrastua, Lorena; San Sebastian, Eider; Quincoces, Ana F; Antony, Claude; Ugalde, Unai

    2003-01-01

    The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae. Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant. Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place. The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes. Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe. The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study. PMID:12435271

  8. 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.

  9. Viral Membrane Fusion and Nucleocapsid Delivery into the Cytoplasm are Distinct Events in Some Flaviviruses

    PubMed Central

    Nour, Adel M.; Li, Yue; Wolenski, Joseph; Modis, Yorgo

    2013-01-01

    Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion. PMID:24039574

  10. Two coiled-coil domains of Chlamydia trachomatis IncA affect membrane fusion events during infection.

    PubMed

    Ronzone, Erik; Paumet, Fabienne

    2013-01-01

    Chlamydia trachomatis replicates in a parasitophorous membrane-bound compartment called an inclusion. The inclusions corrupt host vesicle trafficking networks to avoid the degradative endolysosomal pathway but promote fusion with each other in order to sustain higher bacterial loads in a process known as homotypic fusion. The Chlamydia protein IncA (Inclusion protein A) appears to play central roles in both these processes as it participates to homotypic fusion and inhibits endocytic SNARE-mediated membrane fusion. How IncA selectively inhibits or activates membrane fusion remains poorly understood. In this study, we analyzed the spatial and molecular determinants of IncA's fusogenic and inhibitory functions. Using a cell-free membrane fusion assay, we found that inhibition of SNARE-mediated fusion requires IncA to be on the same membrane as the endocytic SNARE proteins. IncA displays two coiled-coil domains showing high homology with SNARE proteins. Domain swap and deletion experiments revealed that although both these domains are capable of independently inhibiting SNARE-mediated fusion, these two coiled-coil domains cooperate in mediating IncA multimerization and homotypic membrane interaction. Our results support the hypothesis that Chlamydia employs SNARE-like virulence factors that positively and negatively affect membrane fusion and promote infection.

  11. 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

  12. 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

  13. De novo design of conformationally flexible transmembrane peptides driving membrane fusion

    PubMed Central

    Hofmann, Mathias W.; Weise, Katrin; Ollesch, Julian; Agrawal, Prashant; Stalz, Holger; Stelzer, Walter; Hulsbergen, Frans; de Groot, Huub; Gerwert, Klaus; Reed, Jennifer; Langosch, Dieter

    2004-01-01

    Fusion of biological membranes is mediated by distinct integral membrane proteins, e.g., soluble N-ethylmaleimide-sensitive factor attachment protein receptors and viral fusion proteins. Previous work has indicated that the transmembrane segments (TMSs) of such integral membrane proteins play an important role in fusion. Furthermore, peptide mimics of the transmembrane part can drive the fusion of liposomes, and evidence had been obtained that fusogenicity depends on their conformational flexibility. To test this hypothesis, we present a series of unnatural TMSs that were designed de novo based on the structural properties of hydrophobic residues. We find that the fusogenicity of these peptides depends on the ratio of α-helix-promoting Leu and β-sheet-promoting Val residues and is enhanced by helix-destabilizing Pro and Gly residues within their hydrophobic cores. The ability of these peptides to refold from an α-helical state to a β-sheet conformation and backwards was determined under different conditions. Membrane fusogenic peptides with mixed Leu/Val sequences tend to switch more readily between different conformations than a nonfusogenic peptide with an oligo-Leu core. We propose that structural flexibility of these TMSs is a prerequisite of fusogenicity. PMID:15456911

  14. Phylogeny, topology, structure and functions of membrane-bound class III peroxidases in vascular plants.

    PubMed

    Lüthje, Sabine; Meisrimler, Claudia-Nicole; Hopff, David; Möller, Benjamin

    2011-07-01

    Peroxidases are key player in the detoxification of reactive oxygen species during cellular metabolism and oxidative stress. Membrane-bound isoenzymes have been described for peroxidase superfamilies in plants and animals. Recent studies demonstrated a location of peroxidases of the secretory pathway (class III peroxidases) at the tonoplast and the plasma membrane. Proteomic approaches using highly enriched plasma membrane preparations suggest organisation of these peroxidases in microdomains, a developmentally regulation and an induction of isoenzymes by oxidative stress. Phylogenetic relations, topology, putative structures, and physiological function of membrane-bound class III peroxidases will be discussed.

  15. 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.

  16. Interfacial pre-transmembrane domains in viral proteins promoting membrane fusion and fission.

    PubMed

    Lorizate, Maier; Huarte, Nerea; Sáez-Cirión, Asier; Nieva, José L

    2008-01-01

    Membrane fusion and fission underlie two limiting steps of enveloped virus replication cycle: access to the interior of the host-cell (entry) and dissemination of viral progeny after replication (budding), respectively. These dynamic processes proceed mediated by specialized proteins that disrupt and bend the lipid bilayer organization transiently and locally. We introduced Wimley-White membrane-water partitioning free energies of the amino acids as an algorithm for predicting functional domains that may transmit protein conformational energy into membranes. It was found that many viral products possess unusually extended, aromatic-rich pre-transmembrane stretches predicted to stably reside at the membrane interface. Here, we review structure-function studies, as well as data reported on the interaction of representative peptides with model membranes, all of which sustain a functional role for these domains in viral fusion and fission. Since pre-transmembrane sequences also constitute antigenic determinants in a membrane-bound state, we also describe some recent results on their recognition and blocking at membrane interface by neutralizing antibodies.

  17. Modulation of non steroidal anti-inflammatory drug induced membrane fusion by copper coordination of these drugs: anchoring effect.

    PubMed

    Majumdar, Anupa; Chakraborty, Sreeja; Sarkar, Munna

    2014-12-04

    Membrane fusion, an integral event in several biological processes, is characterized by several intermediate steps guided by specific energy barriers. Hence, it requires the aid of fusogens to complete the process. Common fusogens, such as proteins/peptides, have the ability to overcome theses barriers by their conformational reorganization, an advantage not shared by small drug molecules. Hence, drug induced fusion at physiologically relevant drug concentrations is rare and occurs only in the case of the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs). To use drugs to induce and control membrane fusion in various biochemical processes requires the understanding of how different parameters modulate fusion. Also, fusion efficacy needs to be enhanced. Here we have synthesized and used Cu(II) complexes of fusogenic oxicam NSAIDs, Meloxicam and Piroxicam, to induce fusion in model membranes monitored by using DSC, TEM, steady-state, and time-resolved spectroscopy. The ability of the complexes to anchor apposing model membranes to initiate/facilitate fusion has been demonstrated. This results in better fusion efficacy compared to the bare drugs. These complexes can take the fusion to its final step. Unlike other designed membrane anchors, the role of molecular recognition and strength of interaction between molecular partners is obliterated for these preformed Cu(II)-NSAIDs.

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

    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.

  19. 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.

  20. 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.

  1. Membrane Binding by CHMP7 Coordinates ESCRT-III-Dependent Nuclear Envelope Reformation.

    PubMed

    Olmos, Yolanda; Perdrix-Rosell, Anna; Carlton, Jeremy G

    2016-10-10

    In addition to its role in membrane abscission during cytokinesis, viral budding, endosomal sorting, and plasma membrane repair [1], the endosomal sorting complex required for transport-III (ESCRT-III) machinery has recently been shown to seal holes in the reforming nuclear envelope (NE) during mitotic exit [2, 3]. ESCRT-III also acts during interphase to repair the NE upon migration-induced rupture [4, 5], highlighting its key role as an orchestrator of membrane integrity at this organelle. While NE localization of ESCRT-III is dependent upon the ESCRT-III component CHMP7 [3], it is unclear how this complex is able to engage nuclear membranes. Here we show that the N terminus of CHMP7 acts as a novel membrane-binding module. This membrane-binding ability allows CHMP7 to bind to the ER, an organelle continuous with the NE, and it provides a platform to direct NE recruitment of ESCRT-III during mitotic exit. CHMP7's N terminus comprises tandem Winged-Helix domains [6], and, by using homology modeling and structure-function analysis, we identify point mutations that disrupt membrane binding and prevent both ER localization of CHMP7 and its subsequent enrichment at the reforming NE. These mutations also prevent assembly of downstream ESCRT-III components at the reforming NE and proper establishment of post-mitotic nucleo-cytoplasmic compartmentalization. These data identify a novel membrane-binding activity within an ESCRT-III subunit that is essential for post-mitotic nuclear regeneration.

  2. 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

  3. A quantitative model for membrane fusion based on low-energy intermediates

    NASA Astrophysics Data System (ADS)

    Kuzmin, Peter I.; Zimmerberg, Joshua; Chizmadzhev, Yuri A.; Cohen, Fredric S.

    2001-06-01

    The energetics of a fusion pathway is considered, starting from the contact site where two apposed membranes each locally protrude (as "nipples") toward each other. The equilibrium distance between the tips of the two nipples is determined by a balance of physical forces: repulsion caused by hydration and attraction generated by fusion proteins. The energy to create the initial stalk, caused by bending of cis monolayer leaflets, is much less when the stalk forms between nipples rather than parallel flat membranes. The stalk cannot, however, expand by bending deformations alone, because this would necessitate the creation of a hydrophobic void of prohibitively high energy. But small movements of the lipids out of the plane of their monolayers allow transformation of the stalk into a modified stalk. This intermediate, not previously considered, is a low-energy structure that can reconfigure into a fusion pore via an additional intermediate, the prepore. The lipids of this latter structure are oriented as in a fusion pore, but the bilayer is locally compressed. All membrane rearrangements occur in a discrete local region without creation of an extended hemifusion diaphragm. Importantly, all steps of the proposed pathway are energetically feasible.

  4. 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.

  5. Sendai virus-erythrocyte membrane interaction: quantitative and kinetic analysis of viral binding, dissociation, and fusion.

    PubMed

    Hoekstra, D; Klappe, K

    1986-04-01

    A kinetic and quantitative analysis of the binding and fusion of Sendai virus with erythrocyte membranes was performed by using a membrane fusion assay based on the relief of fluorescence self-quenching. At 37 degrees C, the process of virus association displayed a half time of 2.5 min; at 4 degrees C, the half time was 3.0 min. The fraction of the viral dose which became cell associated was independent of the incubation temperature and increased with increasing target membrane concentration. On the average, one erythrocyte ghost can accommodate ca. 1,200 Sendai virus particles. The stability of viral attachment was sensitive to a shift in temperature: a fraction of the virions (ca. 30%), attached at 4 degrees C, rapidly (half time, ca. 2.5 min) eluted from the cell surface at 37 degrees C, irrespective of the presence of free virus in the medium. The elution can be attributed to a spontaneous, temperature-induced release, rather than to viral neuraminidase activity. Competition experiments with nonlabeled virus revealed that viruses destined to fuse do not exchange with free particles in the medium but rather bind in a rapid and irreversible manner. The fusion rate of Sendai virus was affected by the density of the virus particles on the cell surface and became restrained when more than 170 virus particles were attached per ghost. In principle, all virus particles added displayed fusion activity. However, at high virus-to-ghost ratios, only a fraction actually fused, indicating that a limited number of fusion sites exist on the erythrocyte membrane. We estimate that ca. 180 virus particles maximally can fuse with one erythrocyte ghost.

  6. 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.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. 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.

  12. Destabilization of a model membrane by a predicted fusion peptide of fertilin α

    NASA Astrophysics Data System (ADS)

    Schanck, A.; Brasseur, R.; Peuvot, J.

    1998-02-01

    The subunit of the guinea pig fertilin (previously known as PH-30, an integral membrane protein involved in sperm-egg binding and fusion) is predicted to be a potential fusion protein. The structure of this putative fusion protein was analysed by molecular modeling and we have found a peptidic sequence of 17 residues (D83{-P99}) organized in helix that inserts obliquely in lipid phases. The effect of this synthesized peptide was studied on a model membrane by 31P NMR and light scattering. It appears to increase the size of lipid vesicles and induces structural modifications. We interpret these observations as a destabilization of the lipid organization by this peptide because of its tilted insertion in phospholipid layers. This destabilization could favor membrane fusion. La sous-unité α de la fertiline du cochon d'inde (précédemment appelée PH-30, une protéine membranaire impliquée dans la liaison et la fusion ovule-spermatozoïde) est prédite comme étant une protéine de fusion potentielle. Nous avons analysé la structure de cette protéine par modélisation moléculaire et nous avons trouvé une séquence peptidique de 17 résidus (D83 P99) organisée en hélice qui s'insère de façon oblique dans une phase lipidique. L'effet de ce peptide synthétique a été étudié sur membrane modèle par RMN du 31P et par diffusion de la lumière. Il provoque une augmentation de taille de vésicules lipidiques et induit des modifications structurales. Nous interprétons ces observations en termes de déstabilisation de l'organisation lipidique par ce peptide à cause de son insertion oblique dans la couche lipidique. Cette déstabilisation pourrait favoriser la fusion membranaire.

  13. Regulation of yeast ESCRT-III membrane scission activity by the Doa4 ubiquitin hydrolase.

    PubMed

    Johnson, Natalie; West, Matt; Odorizzi, Greg

    2017-03-01

    ESCRT-III executes membrane scission during the budding of intralumenal vesicles (ILVs) at endosomes. The scission mechanism is unknown but appears to be linked to the cycle of assembly and disassembly of ESCRT-III complexes at membranes. Regulating this cycle is therefore expected to be important for determining the timing of ESCRT-III-mediated membrane scission. We show that in Saccharomyces cerevisiae, ESCRT-III complexes are stabilized and ILV membrane scission is delayed by Doa4, which is the ubiquitin hydrolase that deubiquitinates transmembrane proteins sorted as cargoes into ILVs. These results suggest a mechanism to delay ILV budding while cargoes undergo deubiquitination. We further show that deubiquitination of ILV cargoes is inhibited via Doa4 binding to Vps20, which is the subunit of ESCRT-III that initiates assembly of the complex. Current models suggest that ESCRT-III complexes surround ubiquitinated cargoes to trap them at the site of ILV budding while the cargoes undergo deubiquitination. Thus our results also propose a mechanism to prevent the onset of ILV cargo deubiquitination at the initiation of ESCRT-III complex assembly.

  14. Helicobacter pylori TlyA agglutinates liposomes and induces fusion and permeabilization of the liposome membranes.

    PubMed

    Lata, Kusum; Chattopadhyay, Kausik

    2014-06-10

    Helicobacter pylori TlyA is a pore-forming hemolysin with potent cytotoxic activity. To explore the potential membrane-damaging activity of H. pylori TlyA, we have studied its interaction with the synthetic liposome vesicles. In our study, H. pylori TlyA shows a prominent ability to associate with the liposome vesicles without displaying an obligatory requirement for any protein receptor on the liposome membranes. Interaction of TlyA triggers agglutination of the liposome vesicles. Such agglutinating activity of TlyA could also be observed with erythrocytes before the induction of its pore-forming hemolytic activity. In addition to its agglutinating activity against liposomes, TlyA also induces fusion and disruption of the liposome membranes. Altogether, our study highlights novel membrane-damaging properties of H. pylori TlyA that have not been documented previously with any other TlyA family protein.

  15. Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion

    PubMed Central

    Yue, Peng; Zhang, Yubo; Mei, Kunrong; Wang, Shaoxiao; Lesigang, Johannes; Zhu, Yueyao; Dong, Gang; Guo, Wei

    2017-01-01

    The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) constitute the core machinery for membrane fusion during eukaryotic cell vesicular trafficking. However, how the assembly of the SNARE complex is initiated is unknown. Here we report that Sec3, a component of the exocyst complex that mediates vesicle tethering during exocytosis, directly interacts with the t-SNARE protein Sso2. This interaction promotes the formation of an Sso2-Sec9 ‘binary' t-SNARE complex, the early rate-limiting step in SNARE complex assembly, and stimulates membrane fusion. The crystal structure of the Sec3-Sso2 complex suggests that Sec3 binding induces conformational changes of Sso2 that are crucial for the relief of its auto-inhibition. Interestingly, specific disruption of the Sec3–Sso2 interaction in cells blocks exocytosis without affecting the function of Sec3 in vesicle tethering. Our study reveals an activation mechanism for SNARE complex assembly, and uncovers a role of the exocyst in promoting membrane fusion in addition to vesicle tethering. PMID:28112172

  16. III-V/Si hybrid photonic devices by direct fusion bonding

    NASA Astrophysics Data System (ADS)

    Tanabe, Katsuaki; Watanabe, Katsuyuki; Arakawa, Yasuhiko

    2012-04-01

    Monolithic integration of III-V compound semiconductors on silicon is highly sought after for high-speed, low-power-consumption silicon photonics and low-cost, light-weight photovoltaics. Here we present a GaAs/Si direct fusion bonding technique to provide highly conductive and transparent heterojunctions by heterointerfacial band engineering in relation to doping concentrations. Metal- and oxide-free GaAs/Si ohmic heterojunctions have been formed at 300°C sufficiently low to inhibit active material degradation. We have demonstrated 1.3 μm InAs/GaAs quantum dot lasers on Si substrates with the lowest threshold current density of any laser on Si to date, and AlGaAs/Si dual-junction solar cells, by p-GaAs/p-Si and p-GaAs/n-Si bonding, respectively. Our direct semiconductor bonding technique opens up a new pathway for realizing ultrahigh efficiency multijunction solar cells with ideal bandgap combinations that are free from lattice-match restrictions required in conventional heteroepitaxy, as well as enabling the creation of novel high performance and practical optoelectronic devices by III-V/Si hybrid integration.

  17. III-V/Si hybrid photonic devices by direct fusion bonding

    PubMed Central

    Tanabe, Katsuaki; Watanabe, Katsuyuki; Arakawa, Yasuhiko

    2012-01-01

    Monolithic integration of III-V compound semiconductors on silicon is highly sought after for high-speed, low-power-consumption silicon photonics and low-cost, light-weight photovoltaics. Here we present a GaAs/Si direct fusion bonding technique to provide highly conductive and transparent heterojunctions by heterointerfacial band engineering in relation to doping concentrations. Metal- and oxide-free GaAs/Si ohmic heterojunctions have been formed at 300°C; sufficiently low to inhibit active material degradation. We have demonstrated 1.3 μm InAs/GaAs quantum dot lasers on Si substrates with the lowest threshold current density of any laser on Si to date, and AlGaAs/Si dual-junction solar cells, by p-GaAs/p-Si and p-GaAs/n-Si bonding, respectively. Our direct semiconductor bonding technique opens up a new pathway for realizing ultrahigh efficiency multijunction solar cells with ideal bandgap combinations that are free from lattice-match restrictions required in conventional heteroepitaxy, as well as enabling the creation of novel high performance and practical optoelectronic devices by III-V/Si hybrid integration. PMID:22470842

  18. 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.

  19. 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.

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

    PubMed

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

    2015-12-15

    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.

  1. 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

  2. Dynamic light scattering analysis of SNARE-driven membrane fusion and the effects of SNARE-binding flavonoids.

    PubMed

    Yang, Yoosoo; Heo, Paul; Kong, Byoungjae; Park, Jun-Bum; Jung, Young-Hun; Shin, Jonghyeok; Jeong, Cherlhyun; Kweon, Dae-Hyuk

    2015-10-02

    Soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) proteins generate energy required for membrane fusion. They form a parallelly aligned four-helix bundle called the SNARE complex, whose formation is initiated from the N terminus and proceeds toward the membrane-proximal C terminus. Previously, we have shown that this zippering-like process can be controlled by several flavonoids that bind to the intermediate structures formed during the SNARE zippering. Here, our aim was to test whether the fluorescence resonance energy transfer signals that are observed during the inner leaflet mixing assay indeed represent the hemifused vesicles. We show that changes in vesicle size accompanying the merging of bilayers is a good measure of progression of the membrane fusion. Two merging vesicles with the same size D in diameter exhibited their hydrodynamic diameters 2D + d (d, intermembrane distance), 2D and 2D as membrane fusion progressed from vesicle docking to hemifusion and full fusion, respectively. A dynamic light scattering assay of membrane fusion suggested that myricetin stopped membrane fusion at the hemifusion state, whereas delphinidin and cyanidin prevented the docking of the vesicles. These results are consistent with our previous findings in fluorescence resonance energy transfer assays.

  3. Interaction of anionic phenylene ethynylene polymers with lipids: from membrane embedding to liposome fusion.

    PubMed

    Karam, Pierre; Hariri, Amani A; Calver, Christina F; Zhao, Xiaoyong; Schanze, Kirk S; Cosa, Gonzalo

    2014-09-09

    Here we report spectroscopic studies on the interaction of negatively charged, amphiphilic polyphenylene ethynylene (PPE) polymers with liposomes prepared either from negative, positive or zwitterionic lipids. Emission spectra of PPEs of 7 and 49 average repeat units bearing carboxylate terminated side chains showed that the polymer embeds within positively charged lipids where it exists as free chains. No interaction was observed between PPEs and negatively charged lipids. Here the polymer remained aggregated giving rise to broad emission spectra characteristic of the aggregate species. In zwitterionic lipids, we observed that the majority of the polymer remained aggregated yet a small fraction readily embedded within the membrane. Titration experiments revealed that saturation of zwitterionic lipids with polymer typically occurred at a polymer repeat unit to lipid mole ratio close to 0.05. No further membrane embedding was observed above that point. For liposomes prepared from positively charged lipids, saturation was observed at a PPE repeat unit to lipid mole ratio of ∼0.1 and liposome precipitation was observed above this point. FRET studies showed that precipitation was preceded by lipid mixing and liposome fusion induced by the PPEs. This behavior was prominent for the longer polymer and negligible for the shorter polymer at a repeat unit to lipid mole ratio of 0.05. We postulate that fusion is the consequence of membrane destabilization whereby the longer polymer gives rise to more extensive membrane deformation than the shorter polymer.

  4. 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

  5. Membrane permeability transition and dysfunction of rice mitochondria effected by Er(III).

    PubMed

    Gao, Jia-ling; Wu, Man; Wang, Xuan; Zhang, Ye-zhong; Jiang, Feng-lei; Liu, Yi; Dai, Jie

    2015-02-01

    Herein, the biological effects of heavy rare earth ion Er(III) on rice mitochondria were comprehensively investigated mainly by spectroscopic methods. The experimental results demonstrated that Er(III) could lead to the swelling of rice mitochondria, collapse of mitochondrial transmembrane potential, decrease of membrane fluidity, promotion of H(+) permeability and suppression of K(+) permeability. These further indicated that Er(III) could induce the mitochondrial permeability transition (MPT) and the dysfunction of rice mitochondria. The ultra-structure change of mitochondria observed by transmission electron microscopy (TEM) also proved that Er(III) induced MPT. Moreover, the testing results of the protective effect of four different agents on mitochondrial swelling implied that the thiol chelation on the mitochondrial inner membrane was the main reason that caused the MPT.

  6. Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils.

    PubMed Central

    Blackwood, R A; Smolen, J E; Hessler, R J; Harsh, D M; Transue, A

    1996-01-01

    Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide ('DPX') to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 microM, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion. PMID:8670059

  7. Type II integral membrane protein, TM of J paramyxovirus promotes cell-to-cell fusion.

    PubMed

    Li, Zhuo; Hung, Cher; Paterson, Reay G; Michel, Frank; Fuentes, Sandra; Place, Ryan; Lin, Yuan; Hogan, Robert J; Lamb, Robert A; He, Biao

    2015-10-06

    Paramyxoviruses include many important animal and human pathogens. Most paramyxoviruses have two integral membrane proteins: fusion protein (F) and attachment proteins hemagglutinin, hemagglutinin-neuraminidase, or glycoprotein (G), which are critical for viral entry into cells. J paramyxovirus (JPV) encodes four integral membrane proteins: F, G, SH, and transmembrane (TM). The function of TM is not known. In this work, we have generated a viable JPV lacking TM (JPV∆TM). JPV∆TM formed opaque plaques compared with JPV. Quantitative syncytia assays showed that JPV∆TM was defective in promoting cell-to-cell fusion (i.e., syncytia formation) compared with JPV. Furthermore, cells separately expressing F, G, TM, or F plus G did not form syncytia whereas cells expressing F plus TM formed some syncytia. However, syncytia formation was much greater with coexpression of F, G, and TM. Biochemical analysis indicates that F, G, and TM interact with each other. A small hydrophobic region in the TM ectodomain from amino acid residues 118 to 132, the hydrophobic loop (HL), was important for syncytial promotion, suggesting that the TM HL region plays a critical role in cell-to-cell fusion.

  8. 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.

  9. 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.

  10. 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.

  11. 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

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

    DOE PAGES

    McCullough, John; Clippinger, Amy K.; Talledge, Nathaniel; ...

    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

  13. Free energy landscape of rim-pore expansion in membrane fusion.

    PubMed

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

    2014-11-18

    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.

  14. Role of a Putative gp41 Dimerization Domain in Human Immunodeficiency Virus Type 1 Membrane Fusion

    SciTech Connect

    Liu, J.; Deng, Y; Li, Q; Dey, A; Moore, J; Lu, M

    2010-01-01

    The entry of human immunodeficiency virus type 1 (HIV-1) into a target cell entails a series of conformational changes in the gp41 transmembrane glycoprotein that mediates the fusion of the viral and target cell membranes. A trimer-of-hairpins structure formed by the association of two heptad repeat (HR) regions of the gp41 ectodomain has been implicated in a late step of the fusion pathway. Earlier native and intermediate states of the protein are postulated to mediate the antiviral activity of the fusion inhibitor enfuvirtide and of broadly neutralizing monoclonal antibodies (NAbs), but the details of these structures remain unknown. Here, we report the identification and crystal structure of a dimerization domain in the C-terminal ectodomain of gp41 (residues 630 to 683, or C54). Two C54 monomers associate to form an asymmetric, antiparallel coiled coil with two distinct C-terminal {alpha}-helical overhangs. This dimer structure is conferred largely by interactions within a central core that corresponds to the sequence of enfuvirtide. The mutagenic alteration of the dimer interface severely impairs the infectivity of Env-pseudotyped viruses. Moreover, the C54 structure binds tightly to both the 2F5 and 4E10 NAbs and likely represents a potential intermediate conformation of gp41. These results should enhance our understanding of the molecular basis of the gp41 fusogenic structural transitions and thereby guide rational, structure-based efforts to design new fusion inhibitors and vaccine candidates intended to induce broadly neutralizing antibodies.

  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. Real-time analysis of human immunodeficiency virus type 1 Env-mediated membrane fusion by fluorescence resonance energy transfer.

    PubMed

    Furuta, Rika A; Nishikawa, Masao; Fujisawa, Jun-ichi

    2006-02-01

    Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env)-mediated membrane fusion occurs as a sequence of events that is triggered by CD4 binding to the Env gp120 subunit. In this study, we analyzed the dynamics of Env-mediated membrane fusion at the single-cell level using fluorescent fusion proteins and confocal laser fluorescent microscopy. Either enhanced cyan or yellow fluorescent protein (CFP and YFP, respectively) was fused to the end of the cytoplasmic regions of the HIV-1 receptors (CD4 and CCR5) and Env proteins. Real-time imaging of membrane fusion mediated by these recombinant proteins revealed that the kinetics of fusion in our system was faster than that previously reported. Analysis of the receptor interaction by fluorescence resonance energy transfer (FRET) at the single-cell level demonstrated a tendency for oligomerization of CD4-CD4, but not of CD4-CCR5, in the absence of Env-expressing cells. However, when Env-expressing cells attached to the receptor cells, FRET produced by CD4-CCR5 interaction was increased; the FRET intensity began to decline before the formation of the fusion pore. These changes in FRET may represent the temporal association of these receptors, triggered by gp120 binding, and their dissociation during the formation of the fusion pore. In addition, the FRET analysis of receptor interactions in the presence of fusion inhibitors showed that not only inhibitors acting on CCR5 but also the gp41-derived peptide T-20 interfered with CD4-CCR5 interaction during fusion. These data suggest that T-20 could affect the formation of Env-receptors complexes during the membrane fusion.

  17. 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.

  18. Tetraspanins CD9 and CD81 modulate HIV-1-induced membrane fusion.

    PubMed

    Gordón-Alonso, Mónica; Yañez-Mó, María; Barreiro, Olga; Alvarez, Susana; Muñoz-Fernández, M Angeles; Valenzuela-Fernández, Agustín; Sánchez-Madrid, Francisco

    2006-10-15

    Protein organization on the membrane of target cells may modulate HIV-1 transmission. Since the tetraspanin CD81 is associated to CD4, the receptor of HIV-1 envelope protein (Env; gp120/gp41), we have explored the possibility that this molecule may modulate the initial steps of HIV-1 infection. On the other hand, CD81 belongs to the tetraspanin family, which has been described as organizers of protein microdomains on the plasma membrane. Therefore, the role of CD81 and other related tetraspanin, CD9, on the cell-to-cell fusion process mediated by HIV-1 was studied. We found that anti-tetraspanin Abs enhanced the syncytia formation induced by HIV-1 envelope proteins and viral entry in human T lymphoblasts. In addition, anti-CD81 Abs triggered its clustering in patches, where CD4 and CXCR4 were included. Moreover, the knocking down of CD81 and CD9 expression resulted in an increase in syncytia formation and viral entry. Accordingly, overexpression of CD81 and CD9 rendered cells less susceptible to Env-mediated syncytia formation. These data indicate that CD9 and CD81 have an important role in membrane fusion induced by HIV-1 envelope.

  19. 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.

  20. 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.

  1. Molecular and structural basis of ESCRT-III recruitment to membranes during archaeal cell division.

    PubMed

    Samson, Rachel Y; Obita, Takayuki; Hodgson, Ben; Shaw, Michael K; Chong, Parkson Lee-Gau; Williams, Roger L; Bell, Stephen D

    2011-01-21

    Members of the crenarchaeal kingdom, such as Sulfolobus, divide by binary fission yet lack genes for the otherwise near-ubiquitous tubulin and actin superfamilies of cytoskeletal proteins. Recent work has established that Sulfolobus homologs of the eukaryotic ESCRT-III and Vps4 components of the ESCRT machinery play an important role in Sulfolobus cell division. In eukaryotes, several pathways recruit ESCRT-III proteins to their sites of action. However, the positioning determinants for archaeal ESCRT-III are not known. Here, we identify a protein, CdvA, that is responsible for recruiting Sulfolobus ESCRT-III to membranes. Overexpression of the isolated ESCRT-III domain that interacts with CdvA results in the generation of nucleoid-free cells. Furthermore, CdvA and ESCRT-III synergize to deform archaeal membranes in vitro. The structure of the CdvA/ESCRT-III interface gives insight into the evolution of the more complex and modular eukaryotic ESCRT complex.

  2. 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

  3. Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during Sendai virus membrane fusion. II. Kinetics of bacteriophage. lambda. DNA injection

    SciTech Connect

    Novick, S.L.

    1990-01-01

    Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator 3-(trifluoromethyl)-3-(m({sup 125}I) iodophenyl)diazirine. During Sendai virus fusion with liposomes composed of cardiolipin or phosphatidylserine, the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F{sub 1} subunit with the target membrane is an initiating event in fusion. Correlation of hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. The F{sub 1} subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and the F{sub 2} subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions. Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion.

  4. Haploid Genetic Screen Reveals a Profound and Direct Dependence on Cholesterol for Hantavirus Membrane Fusion

    PubMed Central

    Kleinfelter, Lara M.; Jangra, Rohit K.; Jae, Lucas T.; Herbert, Andrew S.; Mittler, Eva; Stiles, Katie M.; Wirchnianski, Ariel S.; Kielian, Margaret; Brummelkamp, Thijn R.

    2015-01-01

    ABSTRACT Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) in the Old World and a highly fatal hantavirus cardiopulmonary syndrome (HCPS) in the New World. No vaccines or antiviral therapies are currently available to prevent or treat hantavirus disease, and gaps in our understanding of how hantaviruses enter cells challenge the search for therapeutics. We performed a haploid genetic screen in human cells to identify host factors required for entry by Andes virus, a highly virulent New World hantavirus. We found that multiple genes involved in cholesterol sensing, regulation, and biosynthesis, including key components of the sterol response element-binding protein (SREBP) pathway, are critical for Andes virus entry. Genetic or pharmacological disruption of the membrane-bound transcription factor peptidase/site-1 protease (MBTPS1/S1P), an SREBP control element, dramatically reduced infection by virulent hantaviruses of both the Old World and New World clades but not by rhabdoviruses or alphaviruses, indicating that this pathway is broadly, but selectively, required by hantaviruses. These results could be fully explained as arising from the modest depletion of cellular membrane cholesterol that accompanied S1P disruption. Mechanistic studies of cells and with protein-free liposomes suggested that high levels of cholesterol are specifically needed for hantavirus membrane fusion. Taken together, our results indicate that the profound dependence on target membrane cholesterol is a fundamental, and unusual, biophysical property of hantavirus glycoprotein-membrane interactions during entry. PMID:26126854

  5. Potential electrostatic interactions in multiple regions affect human metapneumovirus F-mediated membrane fusion.

    PubMed

    Chang, Andres; Hackett, Brent A; Winter, Christine C; Buchholz, Ursula J; Dutch, Rebecca Ellis

    2012-09-01

    The recently identified human metapneumovirus (HMPV) is a worldwide respiratory virus affecting all age groups and causing pneumonia and bronchiolitis in severe cases. Despite its clinical significance, no specific antiviral agents have been approved for treatment of HMPV infection. Unlike the case for most paramyxoviruses, the fusion proteins (F) of a number of strains, including the clinical isolate CAN97-83, can be triggered by low pH. We recently reported that residue H435 in the HRB linker domain acts as a pH sensor for HMPV CAN97-83 F, likely through electrostatic repulsion forces between a protonated H435 and its surrounding basic residues, K295, R396, and K438, at low pH. Through site-directed mutagenesis, we demonstrated that a positive charge at position 435 is required but not sufficient for F-mediated membrane fusion. Arginine or lysine substitution at position 435 resulted in a hyperfusogenic F protein, while replacement with aspartate or glutamate abolished fusion activity. Studies with recombinant viruses carrying mutations in this region confirmed its importance. Furthermore, a second region within the F(2) domain identified as being rich in charged residues was found to modulate fusion activity of HMPV F. Loss of charge at residues E51, D54, and E56 altered local folding and overall stability of the F protein, with dramatic consequences for fusion activity. As a whole, these studies implicate charged residues and potential electrostatic interactions in function, pH sensing, and overall stability of HMPV F.

  6. Obatoclax Inhibits Alphavirus Membrane Fusion by Neutralizing the Acidic Environment of Endocytic Compartments.

    PubMed

    Varghese, Finny S; Rausalu, Kai; Hakanen, Marika; Saul, Sirle; Kümmerer, Beate M; Susi, Petri; Merits, Andres; Ahola, Tero

    2017-03-01

    As new pathogenic viruses continue to emerge, it is paramount to have intervention strategies that target a common denominator in these pathogens. The fusion of viral and cellular membranes during viral entry is one such process that is used by many pathogenic viruses, including chikungunya virus, West Nile virus, and influenza virus. Obatoclax, a small-molecule antagonist of the Bcl-2 family of proteins, was previously determined to have activity against influenza A virus and also Sindbis virus. Here, we report it to be active against alphaviruses, like chikungunya virus (50% effective concentration [EC50] = 0.03 μM) and Semliki Forest virus (SFV; EC50 = 0.11 μM). Obatoclax inhibited viral entry processes in an SFV temperature-sensitive mutant entry assay. A neutral red retention assay revealed that obatoclax induces the rapid neutralization of the acidic environment of endolysosomal vesicles and thereby most likely inhibits viral fusion. Characterization of escape mutants revealed that the L369I mutation in the SFV E1 fusion protein was sufficient to confer partial resistance against obatoclax. Other inhibitors that target the Bcl-2 family of antiapoptotic proteins inhibited neither viral entry nor endolysosomal acidification, suggesting that the antiviral mechanism of obatoclax does not depend on its anticancer targets. Obatoclax inhibited the growth of flaviviruses, like Zika virus, West Nile virus, and yellow fever virus, which require low pH for fusion, but not that of pH-independent picornaviruses, like coxsackievirus A9, echovirus 6, and echovirus 7. In conclusion, obatoclax is a novel inhibitor of endosomal acidification that prevents viral fusion and that could be pursued as a potential broad-spectrum antiviral candidate.

  7. A residue located at the junction of the head and stalk regions of measles virus fusion protein regulates membrane fusion by controlling conformational stability.

    PubMed

    Satoh, Yuto; Yonemori, Saeka; Hirose, Mitsuhiro; Shogaki, Hiroko; Wakimoto, Hiroshi; Kitagawa, Yoshinori; Gotoh, Bin; Shirai, Tsuyoshi; Takahashi, Ken-Ichi; Itoh, Masae

    2017-02-01

    The fusion (F) protein of measles virus performs refolding from the thermodynamically metastable prefusion form to the highly stable postfusion form via an activated unstable intermediate stage, to induce membrane fusion. Some amino acids involved in the fusion regulation cluster in the heptad repeat B (HR-B) domain of the stalk region, among which substitution of residue 465 by various amino acids revealed that fusion activity correlates well with its side chain length from the Cα (P<0.01) and van der Waals volume (P<0.001), except for Phe, Tyr, Trp, Pro and His carrying ring structures. Directed towards the head region, longer side chains of the non-ring-type 465 residues penetrate more deeply into the head region and may disturb the hydrophobic interaction between the stalk and head regions and cause destabilization of the molecule by lowering the energy barrier for refolding, which conferred the F protein enhanced fusion activity. Contrarily, the side chain of ring-type 465 residues turned away from the head region, resulting in not only no contact with the head region but also extensive coverage of the HR-B surface, which may prevent the dissociation of the HR-B bundle for initiation of membrane fusion and suppress fusion activity. Located in the HR-B domain just at the junction between the head and stalk regions, amino acid 465 is endowed with a possible ability to either destabilize or stabilize the F protein depending on its molecular volume and the direction of the side chain, regulating fusion activity of measles virus F protein.

  8. NMR structures and localization of the potential fusion peptides and the pre-transmembrane region of SARS-CoV: Implications in membrane fusion.

    PubMed

    Mahajan, Mukesh; Bhattacharjya, Surajit

    2015-02-01

    Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) poses a serious public health hazard. The S2 subunit of the S glycoprotein of SARS-CoV carries out fusion between the virus and the host cells. However, the exact mechanism of the cell fusion process is not well understood. Current model suggests that a conformational transition, upon receptor recognition, of the two heptad core regions of S2 may expose the hydrophobic fusogenic peptide or fusion peptide for membrane insertion. Three regions of the S2 subunit have been proposed to be involved in cell-cell fusion. The N-terminal fusion peptide (FP, residues 770-788), an internal fusion peptide (IFP, residues 873-888) and the pre-transmembrane region (PTM, residues 1185-1202) demonstrated interactions with model lipid membranes and potentially involved in the fusion process. Here, we have determined atomic resolution structures of these three peptides in DPC detergent micelles by solution NMR. FP assumes α-helical conformation with significant distortion at the central Gly residues; enabling a close packing among sidechains of aromatic residues including W, Y and F. The 3-D structure of PMT is characterized by a helix-loop-helix with extensive aromatic interactions within the helices. IFP adopts a rather straight α-helical conformation defined by packing among sidechains of aromatic and aliphatic residues. Paramagnetic spin labeled NMR has demonstrated surface localization of PMT whereas FP and IFP inserted into the micelles. Collectively, data presented in this study will aid in understanding fusion mechanism of SARS-CoV.

  9. Role for the Terminal Clasp of HIV-1 gp41 Glycoprotein in the Initiation of Membrane Fusion*

    PubMed Central

    Lay, Chan-Sien; Ludlow, Louise E.; Stapleton, David; Bellamy-McIntyre, Anna K.; Ramsland, Paul A.; Drummer, Heidi E.; Poumbourios, Pantelis

    2011-01-01

    The binding by HIV-1 gp120 to CD4 and a chemokine receptor activates the membrane fusion glycoprotein, gp41. The fusion function of gp41 involves the refolding of its core into a 6-helix bundle, which apposes the lipophilic termini (the fusion peptide and transmembrane domain) and the associated cell and viral membranes, leading to their fusion. In this study, we examined the functional role of the polar segment and membrane proximal external region (MPER), which link the fusion peptide and transmembrane domain, respectively, to the core domain and interact to form a terminal clasp adjacent to the core. Limited proteolysis indicated that the terminal clasp is destabilized by simultaneous I535A/V539G mutations within the polar segment and mutations within the MPER. The destabilizing effects of I535A/V539G correlated with defective cell-cell fusion, viral entry, and viral replication. By using lipophilic and cytoplasmic fluorescent dye transfer assays, we found that terminal clasp destabilization is linked to a block in the lipid mixing/hemifusion phase of the membrane fusion cascade. Because the biosynthesis of the prefusion gp120-gp41 complex did not appear to be affected by I535A/V539G, we infer that the hemifusion block is due to a specific effect on the trimer of hairpins conformation of gp41. By contrast, the decreased fusion function of the MPER mutants correlated with a decrease in the interfacial hydropathy of the MPER sequence, suggesting that the prefusion Env complex had been adversely affected in these cases. These findings reveal a novel conserved functional target for the discovery of fusion inhibitors. PMID:21976663

  10. Membrane Fusion Protein Annexin 7: A Common Site of Action for Calcium, Guanosine Triphosphate, Protein Kinase C and Botulinum Toxin Type C in Regulated Exocytosis

    DTIC Science & Technology

    2002-01-01

    approaches to directly test the effects of these agents on annexin 7. Annexin 7 (ANX7) is a calcium-dependent GTP-activated membrane fusion protein. In a...research project, we combined both in vitro and in vivo approaches to directly test the effects of these agents on annexin 7. Annexin 7 (ANX7) is a...exocytotic membrane fusion process. The experimental strategies designed to test this hypothesis include the reconstituted membrane fusion system using

  11. Automatically Identifying Fusion Events between GLUT4 Storage Vesicles and the Plasma Membrane in TIRF Microscopy Image Sequences.

    PubMed

    Wu, Jian; Xu, Yingke; Feng, Zhouyan; Zheng, Xiaoxiang

    2015-01-01

    Quantitative analysis of the dynamic behavior about membrane-bound secretory vesicles has proven to be important in biological research. This paper proposes a novel approach to automatically identify the elusive fusion events between VAMP2-pHluorin labeled GLUT4 storage vesicles (GSVs) and the plasma membrane. The differentiation is implemented to detect the initiation of fusion events by modified forward subtraction of consecutive frames in the TIRFM image sequence. Spatially connected pixels in difference images brighter than a specified adaptive threshold are grouped into a distinct fusion spot. The vesicles are located at the intensity-weighted centroid of their fusion spots. To reveal the true in vivo nature of a fusion event, 2D Gaussian fitting for the fusion spot is used to derive the intensity-weighted centroid and the spot size during the fusion process. The fusion event and its termination can be determined according to the change of spot size. The method is evaluated on real experiment data with ground truth annotated by expert cell biologists. The evaluation results show that it can achieve relatively high accuracy comparing favorably to the manual analysis, yet at a small fraction of time.

  12. Reassessment of the lineage fusion hypothesis for the origin of double membrane bacteria.

    PubMed

    Swithers, Kristen S; Fournier, Gregory P; Green, Anna G; Gogarten, J Peter; Lapierre, Pascal

    2011-01-01

    In 2009, James Lake introduced a new hypothesis in which reticulate phylogeny reconstruction is used to elucidate the origin of gram-negative bacteria (Nature 460: 967-971). The presented data supported the gram-negative bacteria originating from an ancient endosymbiosis between the Actinobacteria and Clostridia. His conclusion was based on a presence-absence analysis of protein families that divided all prokaryotes into five groups: Actinobacteria, Double Membrane bacteria (DM), Clostridia, Archaea and Bacilli. Of these five groups, the DM are by far the largest and most diverse group compared to the other groupings. While the fusion hypothesis for the origin of double membrane bacteria is enticing, we show that the signal supporting an ancient symbiosis is lost when the DM group is broken down into smaller subgroups. We conclude that the signal detected in James Lake's analysis in part results from a systematic artifact due to group size and diversity combined with low levels of horizontal gene transfer.

  13. 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.

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

    PubMed Central

    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

    2015-01-01

    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

  15. Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly.

    PubMed

    Lee, Il-Hyung; Kai, Hiroyuki; Carlson, Lars-Anders; Groves, Jay T; Hurley, James H

    2015-12-29

    The endosomal sorting complexes required for transport (ESCRT) machinery functions in HIV-1 budding, cytokinesis, multivesicular body biogenesis, and other pathways, in the course of which it interacts with concave membrane necks and bud rims. To test the role of membrane shape in regulating ESCRT assembly, we nanofabricated templates for invaginated supported lipid bilayers. The assembly of the core ESCRT-III subunit CHMP4B/Snf7 is preferentially nucleated in the resulting 100-nm-deep membrane concavities. ESCRT-II and CHMP6 accelerate CHMP4B assembly by increasing the concentration of nucleation seeds. Superresolution imaging was used to visualize CHMP4B/Snf7 concentration in a negatively curved annulus at the rim of the invagination. Although Snf7 assemblies nucleate slowly on flat membranes, outward growth onto the flat membrane is efficiently nucleated at invaginations. The nucleation behavior provides a biophysical explanation for the timing of ESCRT-III recruitment and membrane scission in HIV-1 budding.

  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. Tuning the serum persistence of human serum albumin domain III:diabody fusion proteins

    PubMed Central

    Kenanova, Vania E.; Olafsen, Tove; Salazar, Felix B.; Williams, Lawrence E.; Knowles, Scott; Wu, Anna M.

    2010-01-01

    The long circulation persistence of human serum albumin (HSA) is enabled by its domain III (DIII) interaction with the neonatal Fc receptor (FcRn). A protein scaffold based on HSA DIII was designed. To modify the serum half life of the scaffold, residues H535, H510, and H464 were individually mutated to alanine. HSA DIII wild type (WT) and variants were fused to the anti-carcinoembryonic antigen (CEA) T84.66 diabody (Db), radiolabeled with 124I and injected into xenografted athymic mice for serial PET/CT imaging. All proteins targeted the CEA-positive tumor. The mean residence times (MRT) of the proteins, calculated by quantifying blood activity from the PET images, were: Db-DIII WT (56.7 h), H535A (25 h), H510A (20 h), H464A (17 h), compared with Db (2.9 h). Biodistribution confirmed the order of blood clearance from slow to fast: Db-DIII WT > H535A > H510A > H464A > Db with 4.0, 2.0, 1.8, 1.6 and 0.08 %ID/g of remaining blood activity at 51 h, respectively. This study demonstrates that attenuating the DIII–FcRn interaction provides a way of controlling the pharmacokinetics of the entire Db-DIII fusion protein without compromising tumor targeting. H464 appears to be most crucial for FcRn binding (greatest reduction in MRT), followed by H510 and H535. By mutating the DIII scaffold, we can dial serum kinetics for imaging or therapy applications. PMID:20802234

  18. Asymmetric Requirements for a Rab Gtpase and Snare Proteins in Fusion of Copii Vesicles with Acceptor Membranes

    PubMed Central

    Cao, Xiaochun; Barlowe, Charles

    2000-01-01

    Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion. PMID:10747087

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

    PubMed

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

    2016-09-01

    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.

  20. Dissection of the role of the stable signal peptide of the arenavirus envelope glycoprotein in membrane fusion.

    PubMed

    Messina, Emily L; York, Joanne; Nunberg, Jack H

    2012-06-01

    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.

  1. 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.

  2. Binding of Cerebratulus cytolysin A-III to human erythrocyte membranes.

    PubMed

    Blumenthal, K M

    1985-01-10

    Binding of Cerebratulus lacteus cytolysin A-III to intact human erythrocytes and erythrocyte membranes has been investigated. Binding to ghosts is essentially complete within 2.5 min of mixing which is slightly faster than the rate of hemolysis measured with intact cells. Approximately 4 X 10(4) binding sites per cell, exhibiting a K 0.5 of 0.7 microM exist; this compares with 50% hematocrit of about 0.3 microM for A-III. Binding is absent in ghosts extracted with Nonidet P-40, but is unaffected by pretreatment of ghosts with either trypsin or elastase.

  3. Sensor fusion III; Proceedings of the Meeting, Orlando, FL, Apr. 19, 20, 1990

    SciTech Connect

    Harney, R.C.

    1990-01-01

    Multisensor image segmentation algorithms, scene descriptions from radar and infrared images, and combination of evidences with dependency information in distributed sensor systems are discussed. Preference voting for sensor fusion, a simulation-based test bed for data-association algorithms, receiver operating characteristics for various sensor fusion schemes, and adaptive multisensor change detection are analyzed. A tree-structured sensor fusion architecture for distributed sensor networks and performance modeling of multisensor networks is covered, along with components for sensor fusion systems, including high-precision line-of-sight stabilization for a 1-m space telescope with onboard image processing. The continuous mode of operation of a space-time light modulator is addressed, as well as an embedded knowledge-based system for automatic target recognition and multisensor data fusion for mine detection.

  4. 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

  5. Use of gene fusions to study outer membrane protein localization in Escherichia coli.

    PubMed Central

    Silhavy, T J; Shuman, H A; Beckwith, J; Schwartz, M

    1977-01-01

    Escherichia coli strains have been isolated that produce hybrid proteins comprised of an NH2-terminal sequence from the lamB gene product (an outer membrane protein) and a major portion of the COOH-terminal sequence of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23; a cytoplasmic protein). These proteins exhibit beta-galactosidase activity. One such strain, pop 3105, produces a hybrid protein containing very little of the lamB gene protein; the protein is found in the cytoplasm. The protein found in a second strain, pop 3186, contains much more of the lamB gene protein; a substantial fraction of the beta-galactosidase activity is found in the outer membrane, probably facing outward. These results indicate that information necessary to direct the lamB gene product to its outer membrane location is located within the lamB gene itself. The properties of such fusion strains open up the prospect of a precise genetic analysis of the genetic components involved in protein transport. Images PMID:414221

  6. Planar Supported Membranes with Mobile SNARE Proteins and Quantitative Fluorescence Microscopy Assays to Study Synaptic Vesicle Fusion

    PubMed Central

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

    2017-01-01

    Synaptic vesicle membrane fusion, the process by which neurotransmitter gets released at the presynaptic membrane is mediated by a complex interplay between proteins and lipids. The realization that the lipid bilayer is not just a passive environment where other molecular players like SNARE proteins act, but is itself actively involved in the process, makes the development of biochemical and biophysical assays particularly challenging. We summarize in vitro assays that use planar supported membranes and fluorescence microscopy to address some of the open questions regarding the molecular mechanisms of SNARE-mediated membrane fusion. Most of the assays discussed in this mini-review were developed in our lab over the last 15 years. We emphasize the sample requirements that we found are important for the successful application of these methods. PMID:28360838

  7. Membrane fusion inducers, chloroquine and spermidine increase lipoplex-mediated gene transfection

    SciTech Connect

    Wong-Baeza, Carlos; Bustos, Israel; Serna, Manuel; Tescucano, Alonso; Alcantara-Farfan, Veronica; Ibanez, Miguel; Montanez, Cecilia; Wong, Carlos; Baeza, Isabel

    2010-05-28

    Gene transfection into mammalian cells can be achieved with viral and non-viral vectors. Non-viral vectors, such as cationic lipids that form lipoplexes with DNA, are safer and more stable than viral vectors, but their transfection efficiencies are lower. Here we describe that the simultaneous treatment with a membrane fusion inducer (chlorpromazine or procainamide) plus the lysosomotropic agent chloroquine increases lipoplex-mediated gene transfection in human (HEK293 and C-33 A) and rat (PC12) cell lines (up to 9.2-fold), as well as in situ in BALB/c mice spleens and livers (up to 6-fold); and that the polyamine spermidine increases lipoplex-mediated gene transfection and expression in cell cultures. The use of these four drugs provides a novel, safe and relatively inexpensive way to considerably increase lipoplex-mediated gene transfection efficiency.

  8. 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

  9. Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion

    PubMed Central

    Li, Feng; Tiwari, Neeraj; Rothman, James E.; Pincet, Frederic

    2016-01-01

    Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state. PMID:27601655

  10. Acid phosphatase 2 (ACP2) is required for membrane fusion during influenza virus entry

    PubMed Central

    Lee, Jihye; Kim, Jinhee; Son, Kidong; d’Alexandry d’Orengiani, Anne-Laure Pham Humg; Min, Ji-Young

    2017-01-01

    Influenza viruses exploit host factors to successfully replicate in infected cells. Using small interfering RNA (siRNA) technology, we identified six human genes required for influenza A virus (IAV) replication. Here we focused on the role of acid phosphatase 2 (ACP2), as its knockdown showed the greatest inhibition of IAV replication. In IAV-infected cells, depletion of ACP2 resulted in a significant reduction in the expression of viral proteins and mRNA, and led to the attenuation of virus multi-cycle growth. ACP2 knockdown also decreased replication of seasonal influenza A and B viruses and avian IAVs of the H7 subtype. Interestingly, ACP2 depletion had no effect on the replication of Ebola or hepatitis C virus. Because ACP2 is known to be a lysosomal acid phosphatase, we assessed the role of ACP2 in influenza virus entry. While neither binding of the viral particle to the cell surface nor endosomal acidification was affected in ACP2-depleted cells, fusion of the endosomal and viral membranes was impaired. As a result, downstream steps in viral entry were blocked, including nucleocapsid uncoating and nuclear import of viral ribonucleoproteins. Our results established ACP2 as a necessary host factor for regulating the fusion step of influenza virus entry. PMID:28272419

  11. Myristoylation of the Arenavirus Envelope Glycoprotein Stable Signal Peptide Is Critical for Membrane Fusion but Dispensable for Virion Morphogenesis

    PubMed Central

    York, Joanne

    2016-01-01

    ABSTRACT Arenaviruses are responsible for severe and often fatal hemorrhagic disease. In the absence of effective antiviral therapies and vaccines, these viruses pose serious threats to public health and biodefense. Arenaviruses enter the host cell by fusion of the viral and endosomal membranes, a process mediated by the virus envelope glycoprotein GPC. Unlike other class I viral fusion proteins, GPC retains its stable signal peptide (SSP) as an essential third subunit in the mature complex. SSP spans the membrane twice and is myristoylated at its cytoplasmic N terminus. Mutations that abolish SSP myristoylation have been shown to reduce pH-induced cell-cell fusion activity of ectopically expressed GPC to ∼20% of wild-type levels. In order to examine the role of SSP myristoylation in the context of the intact virus, we used reverse genetics to generate Junín viruses (Candid #1 isolate) in which the critical glycine-2 residue in SSP was either replaced by alanine (G2A) or deleted (ΔG2). These mutant viruses produced smaller foci of infection in Vero cells and showed an ∼5-fold reduction in specific infectivity, commensurate with the defect in cell-cell fusion. However, virus assembly and GPC incorporation into budded virions were unaffected. Our findings suggest that the myristate moiety is cryptically disposed in the prefusion GPC complex and may function late in the fusion process to promote merging of the viral and cellular membranes. IMPORTANCE Hemorrhagic fever arenaviruses pose significant threats to public health and biodefense. Arenavirus entry into the host cell is promoted by the virus envelope glycoprotein GPC. Unlike other viral envelope glycoproteins, GPC contains a myristoylated stable signal peptide (SSP) as an essential third subunit. Myristoylation has been shown to be important for the membrane fusion activity of recombinantly expressed GPC. Here, we use reverse genetics to study the role of SSP myristoylation in the context of the intact

  12. 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.

  13. The influenza fusion peptide promotes lipid polar head intrusion through hydrogen bonding with phosphates and N-terminal membrane insertion depth.

    PubMed

    Légaré, Sébastien; Lagüe, Patrick

    2014-09-01

    Influenza infection requires fusion between the virus envelope and a host cell endosomal membrane. The influenza hemagglutinin fusion peptide (FP) is essential to viral membrane fusion. It was recently proposed that FPs would fuse membranes by increasing lipid tail protrusion, a membrane fusion transition state. The details of how FPs induce lipid tail protrusion, however, remain to be elucidated. To decipher the molecular mechanism by which FPs promote lipid tail protrusion, we performed molecular dynamics simulations of the wild-type (WT) FP, fusogenic mutant F9A, and nonfusogenic mutant W14A in model bilayers. This article presents the peptide-lipid interaction responsible for lipid tail protrusion and a related lipid perturbation, polar head intrusion, where polar heads are sunk under the membrane surface. The backbone amides from the four N-terminal peptide residues, deeply inserted in the membrane, promoted both perturbations through H bonding with lipid phosphates. Polar head intrusion correlated with peptides N-terminal insertion depth and activity: the N-termini of WT and F9A were inserted deeper into the membrane than nonfusogenic W14A. Based on these results, we propose that FP-induced polar head intrusion would complement lipid tail protrusion in catalyzing membrane fusion by reducing repulsions between juxtaposed membranes headgroups. The presented model provides a framework for further research on membrane fusion and influenza antivirals.

  14. 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.

  15. Reassessment of the Lineage Fusion Hypothesis for the Origin of Double Membrane Bacteria

    PubMed Central

    Swithers, Kristen S.; Fournier, Gregory P.; Green, Anna G.; Gogarten, J. Peter; Lapierre, Pascal

    2011-01-01

    In 2009, James Lake introduced a new hypothesis in which reticulate phylogeny reconstruction is used to elucidate the origin of Gram-negative bacteria (Nature 460: 967–971). The presented data supported the Gram-negative bacteria originating from an ancient endosymbiosis between the Actinobacteria and Clostridia. His conclusion was based on a presence-absence analysis of protein families that divided all prokaryotes into five groups: Actinobacteria, Double Membrane bacteria (DM), Clostridia, Archaea and Bacilli. Of these five groups, the DM are by far the largest and most diverse group compared to the other groupings. While the fusion hypothesis for the origin of double membrane bacteria is enticing, we show that the signal supporting an ancient symbiosis is lost when the DM group is broken down into smaller subgroups. We conclude that the signal detected in James Lake's analysis in part results from a systematic artifact due to group size and diversity combined with low levels of horizontal gene transfer. PMID:21876769

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

    NASA Astrophysics Data System (ADS)

    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.

    2015-02-01

    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 (P2 and P4) 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 P4 mode due to growth of a feature seeded by the tent, which can explain prior inconsistencies between the in-flight P4 and core P4, 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.

  17. DNA Release from Lipoplexes by Anionic Lipids: Correlation with Lipid Mesomorphism, Interfacial Curvature, and Membrane Fusion

    PubMed Central

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

    2004-01-01

    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. PMID:15298910

  18. 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

  19. Crystal Structure of the Membrane Fusion Protein CusB from Escherichia coli

    SciTech Connect

    Su, Chih-Chia; Yang, Feng; Long, Feng; Reyon, Deepak; Routh, Mathew D.; Kuo, Dennis W.; Mokhtari, Adam K.; Van Ornam, Jonathan D.; Rabe, Katherine L.; Hoy, Julie A.; Lee, Young Jin; Rajashankar, Kanagalaghatta R.; Yu, Edward W.

    2010-03-29

    Gram-negative bacteria, such as Escherichia coli, frequently utilize tripartite efflux complexes belonging to the resistance-nodulation-division family to expel diverse toxic compounds from the cell. These systems contain a periplasmic membrane fusion protein (MFP) that is critical for substrate transport. We here present the x-ray structures of the CusB MFP from the copper/silver efflux system of E. coli. This is the first structure of any MFPs associated with heavy-metal efflux transporters. CusB bridges the inner-membrane efflux pump CusA and outer-membrane channel CusC to mediate resistance to Cu{sup +} and Ag{sup +} ions. Two distinct structures of the elongated molecules of CusB were found in the asymmetric unit of a single crystal, which suggests the flexible nature of this protein. Each protomer of CusB can be divided into four different domains, whereby the first three domains are mostly {beta}-strands and the last domain adopts an entirely helical architecture. Unlike other known structures of MFPs, the {alpha}-helical domain of CusB is folded into a three-helix bundle. This three-helix bundle presumably interacts with the periplasmic domain of CusC. The N- and C-termini of CusB form the first {beta}-strand domain, which is found to interact with the periplasmic domain of the CusA efflux pump. Atomic details of how this efflux protein binds Cu{sup +} and Ag{sup +} were revealed by the crystals of the CusB-Cu(I) and CusB-Ag(I) complexes. The structures indicate that CusB consists of multiple binding sites for these metal ions. These findings reveal novel structural features of an MFP in the resistance-nodulation-division efflux system and provide direct evidence that this protein specifically interacts with transported substrates.

  20. 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 Central

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

    2015-01-01

    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. PMID:26297995

  1. 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

  2. Atomic data for controlled fusion research. Volume III. Particle interactions with surfaces

    SciTech Connect

    Thomas, E.W.

    1985-02-01

    This report provides a handbook of data concerning particle solid interactions that are relevant to plasma-wall interactions in fusion devices. Published data have been collected, assessed, and represented by a single functional relationship which is presented in both tabular and graphical form. Mechanisms reviewed here include sputtering, secondary electron emission, particle reflection, and trapping.

  3. Magnesium-induced lipid bilayer microdomain reorganizations: implications for membrane fusion.

    PubMed

    Schultz, Zachary D; Pazos, Ileana M; McNeil-Watson, Fraser K; Lewis, E Neil; Levin, Ira W

    2009-07-23

    Interactions between dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS), combined both as binary lipid bilayer assemblies and separately, under the influence of divalent Mg2+, a membrane bilayer fusogenic agent, are reported. Infrared vibrational spectroscopic analyses of the lipid acyl chain methylene symmetric stretching modes indicate that aggregates of the two phospholipid components exist as domains heterogeneously distributed throughout the binary bilayer system. In the presence of Mg2+, DPPS maintains an ordered orthorhombic subcell gel phase structure through the phase transition temperature, while the DPPC component is only minimally perturbed with respect to the gel to liquid crystalline phase change. The addition of Mg2+ induces a reorganization of the lipid domains in which the gel phase acyl chain planes rearrange from a hexagonal configuration toward a triclinic, parallel chain subcell. Examination of the acyl chain methylene deformation modes at low temperatures allows a determination of DPPS microdomain sizes, which decrease upon the addition of DPPC-d62 in the absence of Mg2+. On adding Mg2+, a uniform DPPS domain size is observed in the binary mixtures. In either the presence or absence of Mg2+, DPPC-d62 aggregates remain in a configuration for which microdomain sizes are not spectroscopically measurable. Analysis of the acyl chain methylene deformation modes for DPPC-d62 in the binary system suggests that clusters of the deuterated lipids are distributed throughout the DPPS matrix. Light scattering and fluorescence measurements indicate that Mg2+ induces both the aggregation and the fusion of the lipid assemblies as a function of the ratio of DPPS to DPPC. The structural reorganizations of the lipid microdomains within the DPPS-DPPC bilayer are interpreted in the context of current concepts regarding lipid bilayer fusion.

  4. 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.

  5. 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

  6. 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.

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

    PubMed

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

    2015-06-02

    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.

  8. A Novel Site of Action for α-SNAP in the SNARE Conformational Cycle Controlling Membrane Fusion

    PubMed Central

    Barszczewski, Marcin; Chua, John J.; Stein, Alexander; Winter, Ulrike; Heintzmann, Rainer; Zilly, Felipe E.; Fasshauer, Dirk; Lang, Thorsten

    2008-01-01

    Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of α-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of α-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of α-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of α-SNAP potently inhibits Ca2+-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an α-SNAP mutant defective in NSF activation is used. We conclude that α-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for α-SNAP in the SNARE cycle that drives exocytotic membrane fusion. PMID:18094056

  9. A novel site of action for alpha-SNAP in the SNARE conformational cycle controlling membrane fusion.

    PubMed

    Barszczewski, Marcin; Chua, John J; Stein, Alexander; Winter, Ulrike; Heintzmann, Rainer; Zilly, Felipe E; Fasshauer, Dirk; Lang, Thorsten; Jahn, Reinhard

    2008-03-01

    Regulated exocytosis in neurons and neuroendocrine cells requires the formation of a stable soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of synaptobrevin-2/vesicle-associated membrane protein 2, synaptosome-associated protein of 25 kDa (SNAP-25), and syntaxin 1. This complex is subsequently disassembled by the concerted action of alpha-SNAP and the ATPases associated with different cellular activities-ATPase N-ethylmaleimide-sensitive factor (NSF). We report that NSF inhibition causes accumulation of alpha-SNAP in clusters on plasma membranes. Clustering is mediated by the binding of alpha-SNAP to uncomplexed syntaxin, because cleavage of syntaxin with botulinum neurotoxin C1 or competition by using antibodies against syntaxin SNARE motif abolishes clustering. Binding of alpha-SNAP potently inhibits Ca(2+)-dependent exocytosis of secretory granules and SNARE-mediated liposome fusion. Membrane clustering and inhibition of both exocytosis and liposome fusion are counteracted by NSF but not when an alpha-SNAP mutant defective in NSF activation is used. We conclude that alpha-SNAP inhibits exocytosis by binding to the syntaxin SNARE motif and in turn prevents SNARE assembly, revealing an unexpected site of action for alpha-SNAP in the SNARE cycle that drives exocytotic membrane fusion.

  10. Ovine Herpesvirus 2 Glycoproteins B, H, and L Are Sufficient for, and Viral Glycoprotein Ov8 Can Enhance, Cell-Cell Membrane Fusion.

    PubMed

    AlHajri, Salim M; Cunha, Cristina W; Nicola, Anthony V; Aguilar, Hector C; Li, Hong; Taus, Naomi S

    2017-03-15

    Ovine herpesvirus 2 (OvHV-2) is a gammaherpesvirus in the genus Macavirus that is carried asymptomatically by sheep. Infection of poorly adapted animals with OvHV-2 results in sheep-associated malignant catarrhal fever, a fatal disease characterized by lymphoproliferation and vasculitis. There is no treatment or vaccine for the disease and no cell culture system to propagate the virus. The lack of cell culture has hindered studies of OvHV-2 biology, including its entry mechanism. As an alternative method to study OvHV-2 glycoproteins responsible for membrane fusion as a part of the entry mechanism, we developed a virus-free cell-to-cell membrane fusion assay to identify the minimum required OvHV-2 glycoproteins to induce membrane fusion. OvHV-2 glycoproteins B, H, and L (gB, gH, and gL) were able to induce membrane fusion together but not when expressed individually. Additionally, open reading frame Ov8, unique to OvHV-2, was found to encode a transmembrane glycoprotein that can significantly enhance membrane fusion. Thus, OvHV-2 gB, gH, and gL are sufficient to induce membrane fusion, while glycoprotein Ov8 plays an enhancing role by an unknown mechanism.IMPORTANCE Herpesviruses enter cells via attachment of the virion to the cellular surface and fusion of the viral envelope with cellular membranes. Virus-cell membrane fusion is an important step for a successful viral infection. Elucidating the roles of viral glycoproteins responsible for membrane fusion is critical toward understanding viral entry. Entry of ovine herpesvirus 2 (OvHV-2), the causative agent of sheep associated-malignant catarrhal fever, which is one of the leading causes of death in bison and other ungulates, has not been well studied due to the lack of a cell culture system to propagate the virus. The identification of OvHV-2 glycoproteins that mediate membrane fusion may help identify viral and/or cellular factors involved in OvHV-2 cell tropism and will advance investigation of cellular

  11. Coordinated binding of Vps4 to ESCRT-III drives membrane neck constriction during MVB vesicle formation.

    PubMed

    Adell, Manuel Alonso Y; Vogel, Georg F; Pakdel, Mehrshad; Müller, Martin; Lindner, Herbert; Hess, Michael W; Teis, David

    2014-04-14

    Five endosomal sorting complexes required for transport (ESCRTs) mediate the degradation of ubiquitinated membrane proteins via multivesicular bodies (MVBs) in lysosomes. ESCRT-0, -I, and -II interact with cargo on endosomes. ESCRT-II also initiates the assembly of a ringlike ESCRT-III filament consisting of Vps20, Snf7, Vps24, and Vps2. The AAA-adenosine triphosphatase Vps4 disassembles and recycles the ESCRT-III complex, thereby terminating the ESCRT pathway. A mechanistic role for Vps4 in intraluminal vesicle (ILV) formation has been unclear. By combining yeast genetics, biochemistry, and electron tomography, we find that ESCRT-III assembly on endosomes is required to induce or stabilize the necks of growing MVB ILVs. Yet, ESCRT-III alone is not sufficient to complete ILV biogenesis. Rather, binding of Vps4 to ESCRT-III, coordinated by interactions with Vps2 and Snf7, is coupled to membrane neck constriction during ILV formation. Thus, Vps4 not only recycles ESCRT-III subunits but also cooperates with ESCRT-III to drive distinct membrane-remodeling steps, which lead to efficient membrane scission at the end of ILV biogenesis in vivo.

  12. 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.

  13. [Development of the MITO-porter, a nano device for mitochondrial drug delivery via membrane fusion].

    PubMed

    Yamada, Yuma

    2014-01-01

    Many human diseases have been reported to be associated with mitochondrial dysfunction. Therefore, mitochondrial therapy would be expected to be useful and productive in the treatment of various diseases. To achieve such an innovative therapy, it will be necessary to deliver therapeutic agents into mitochondria. However, only a limited number of methods are available for accomplishing this. We previously developed the MITO-Porter, a liposome-based carrier that permits macromolecular cargos to be transported into mitochondria via membrane fusion. Intracellular observations using the green fluorescence protein as a model macromolecule confirmed the mitochondrial delivery of a macromolecule by the MITO-Porter. Moreover, when we attempted the mitochondrial delivery of bongkrekic acid (BKA), an antiapoptosis agent, the MITO-Porter enhanced the antiapoptosis effect compared with naked BKA. To construct a device with enhanced performance, the MITO-Porter was coated with cell membrane-fusogenic outer envelopes to produce the dual function (DF)-MITO-Porter. Intracellular observations indicated that the DF-MITO-Porter was more effective in delivering exogenous macromolecules into mitochondria than the conventional MITO-Porter. Furthermore, when biomacromolecules were delivered using the DF-MITO-Porter to estimate the mitochondrial gene targeting of the carrier, the results confirmed that the MITO-Porter system has the potential for use in therapies aimed at mitochondrial DNA. This paper sumarizes our findings on mitochondrial drug delivery systems that are directed toward mitochondrial medicine development and mitochondrial gene therapy. It is expected that the MITO-Porter system will open new research areas in mitochondrial drug delivery systems and have a significant impact on the medical and life sciences.

  14. Ribozyme Targeting the Novel Fusion Junction of EGFRvIII in Breast Cancer

    DTIC Science & Technology

    2003-07-01

    targeting the novel junction of EGFRvyII. * Demonstrate the therapeutic efficacy of an anti-EGFRvIll hammerhead ribozyme targeting the endogenous...first demonstration of the therapeutic efficacy of an anti-EGFRvlII hammerhead ribozyme targeting the endogenous EGFRvAII expression against human...202-687-7505.designed and generated a tumor specific hammerhead ribozyme E-mail: Tangc@georgetown.edu targeted to the novel fusion junction of

  15. The Highly Conserved Proline at Position 438 in Pseudorabies Virus gH Is Important for Regulation of Membrane Fusion

    PubMed Central

    Schröter, Christina; Klupp, Barbara G.; Fuchs, Walter; Gerhard, Marika; Backovic, Marija; Rey, Felix A.

    2014-01-01

    ABSTRACT Membrane fusion in herpesviruses requires viral glycoproteins (g) gB and gH/gL. While gB is considered the actual fusion protein but is nonfusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-proline-cysteine corresponding to positions 437 to 439 in pseudorabies virus (PrV) gH. The PrV-gH structure shows that proline438 induces bending at the end of an alpha-helix, thereby placing cysteine404 and cysteine439 in juxtaposition to allow formation of a strictly conserved disulfide bond. However, PrV vaccine strain Bartha unexpectedly carries a serine at this conserved position. To test the influence of this substitution, we constructed different gH chimeras carrying proline or serine at position 438 in gH derived from either PrV strain Kaplan or strain Bartha. Mutants expressing gH with serine438 showed reduced fusion activity in transient-fusion assays and during infection, with delayed penetration kinetics and a small-plaque phenotype which indicates that proline438 is important for efficient fusion. A more drastic effect was observed when disulfide bond formation was completely blocked by mutation of cysteine404 to serine. Although PrV expressing gHC404S was viable, plaque size and penetration kinetics were drastically reduced. Alteration of serine438 to proline in gH of strain Bartha enhanced cell-to-cell spread and penetration kinetics, but restoration of full activity required additional alteration of aspartic acid to valine at position 59. IMPORTANCE The role of the gH/gL complex in herpesvirus membrane fusion is still unclear. Structural studies predicted a critical role for proline438 in PrV gH to allow the formation of a conserved disulfide bond and correct protein folding. Functional analyses within this study corroborated these structural predictions

  16. DsbA directs efficient expression of outer membrane secretin EscC of the enteropathogenic Escherichia coli type III secretion apparatus.

    PubMed

    Miki, Tsuyoshi; Okada, Nobuhiko; Kim, Yeongsuk; Abe, Akio; Danbara, Hirofumi

    2008-02-01

    The formation of disulfide bond is essential for the folding, activity, and stability of many secreted proteins of Gram-negative bacteria. The disulfide oxidoreductase, DsbA, introduces disulfide bonds into exported proteins from the cytoplasm. In pathogenic bacteria, DsbA is required to process virulence determinants for their folding and assembly. In this study, we investigated the role of DsbA in enteropathogenic Escherichia coli. Here, we show that the DsbA is required for stable expression of outer membrane secretin EscC. DsbA has no effect on LEE transcription as measured with LEE-lacZ fusions. Replacement of either cysteine residue 136 or 155 of EscC with a serine resulted in reduced level of EscC, similar to the effect of the dsbA mutation. These results demonstrate the role of DsbA in assembly of the type III secretion apparatus.

  17. 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.

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

    PubMed Central

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

    2016-01-01

    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

  19. Three conserved C-terminal residues of influenza fusion peptide alter its behavior at the membrane interface.

    PubMed

    Worch, Remigiusz; Krupa, Joanna; Filipek, Alicja; Szymaniec, Anna; Setny, Piotr

    2017-02-01

    The N-terminal fragment of the viral hemagglutinin HA2 subunit is termed a fusion peptide (HAfp). The 23-amino acid peptide (HAfp1-23) contains three C-terminal W21-Y22-G23 residues which are highly conserved among serotypes of influenza A and has been shown to form a tight helical hairpin very distinct from the boomerang structure of HAfp1-20. We studied the effect of peptide length on fusion properties, structural dynamics, and binding to the membrane interface. We developed a novel fusion visualization assay based on FLIM microscopy on giant unilamellar vesicles (GUV). By means of molecular dynamics simulations and spectroscopic measurements, we show that the presence of the three C-terminal W21-Y22-G23 residues promotes the hairpin formation, which orients perpendicularly to the membrane plane and induces more disorder in the surrounding lipids than the less structured HAfp1-20. Moreover, we report cholesterol-enriched domain formation induced exclusively by the longer fusion peptide.

  20. Selective Fusion of Heterogeneous Classifiers for Predicting Substrates of Membrane Transporters.

    PubMed

    Shaikh, Naeem; Sharma, Mahesh; Garg, Prabha

    2017-03-27

    Membrane transporters play a crucial role in determining fate of administered drugs in a biological system. Early identification of plausible transporters for a drug molecule can provide insights into its therapeutic, pharmacokinetic, and toxicological profiles. In the present study, predictive models for classifying small molecules into substrates and nonsubstrates of various pharmaceutically important membrane transporters were developed using quantitative structure-activity relationship (QSAR) and proteochemometric (PCM) approaches. For this purpose, 4575 substrate interactions for these transporters were collected from the Metabolism and Transport Database (Metrabase) and the literature. The transporters selected for this study include (i) six efflux transporters, viz., breast cancer resistance protein (BCRP/ABCG2), P-glycoprotein (P-gp/MDR1), and multidrug resistance proteins (MRP1, MRP2, MRP3, and MRP4), and (ii) seven influx transporters, viz., organic cation transporter (OCT1/SO22A1), peptide transporter (PEPT1/SO15A1), apical sodium-bile acid transporter (ASBT/NTCP2), and organic anion transporting peptides (OATP1A2/SO1A2, OATP1B/SO1B1, OATP1B3/SO1B3, and OATP2B1/SO2B1). Various types of descriptors and machine learning methods (classifiers) were evaluated for the development of robust predictive models. Additionally, ensemble models were developed by bagging of homogeneous classifiers and selective fusion of heterogeneous classifiers. It was observed that the latter approach improves the accuracy of substrate/nonsubstrate prediction for transporters (average correct classification rate of more than 0.80 for external validation). Moreover, structural fragments important in determining the substrate specificity across the various transporters were identified. To demonstrate these fragments on the query molecule, contour maps were generated. The prediction efficacy of the developed models was illustrated by a good correlation between the reported logBB value

  1. The construction and characterization of Neisseria gonorrhoeae lacking protein III in its outer membrane

    PubMed Central

    1989-01-01

    Protein III (PIII) is a highly conserved, antigenically stable gonococcal outer membrane protein that is closely associated with the major outer membrane protein, protein I (PI). We have previously reported the cloning of the PIII gene. This gene was inserted into the Eco RI site of the runaway plasmid pMOB45. The beta-lactamase (beta la) Bam HI restriction fragment from the gonococcal plasmid pFA3 was inserted at the Xba I site in the PIII gene. The plasmid construct was Hae III methylated and the PIII/beta la insert was excised with Eco RI and used to transform gonococcal strain F62. One beta la+, ampicillin- resistant transformant was isolated and designated 2D. A Western blot of 2D whole cell lysate was probed with affinity-purified polyclonal PIII antisera. No PIII reactivity was detected. Southern blot analysis was performed on F62 and 2D chromosomal DNA that were cut with Eco RI or Cla I. A PIII DNA probe hybridized with fragments 2.2 kb larger in strain 2D than strain F62. This corresponds to the size of the beta la insert. A beta la-specific probe hybridized with the same 2D restriction fragments as above, but did not react with any F62 fragments, confirming that homologous recombination had occurred. There were minimal phenotypic changes between 2D and its parent strain, F62. Chromosomal DNA from 2D was able to transform gonococcal strains F62, UU1, and Pgh 3-2, rendering these PIII-. 2D and other PIII- transformants can now be used to study the role of PIII in gonococcal physiology, metabolism, membrane structure, and pathogenesis. Moreover, we now have organisms from which we can purify gonococcal proteins without PIII contamination. PMID:2499656

  2. In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.

    PubMed Central

    Cartwright, C P; Tipper, D J

    1991-01-01

    Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells. Images PMID:2017168

  3. 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.

  4. Proteolytic Cleavage of the Fusion Protein but Not Membrane Fusion Is Required for Measles Virus-Induced Immunosuppression In Vitro

    PubMed Central

    Weidmann, Armin; Maisner, Andrea; Garten, Wolfgang; Seufert, Marion; ter Meulen, Volker; Schneider-Schaulies, Sibylle

    2000-01-01

    Immunosuppression induced by measles virus (MV) is associated with unresponsiveness of peripheral blood lymphocytes (PBL) to mitogenic stimulation ex vivo and in vitro. In mixed lymphocyte cultures and in an experimental animal model, the expression of the MV glycoproteins on the surface of UV-inactivated MV particles, MV-infected cells, or cells transfected to coexpress the MV fusion (F) and the hemagglutinin (H) proteins was found to be necessary and sufficient for this phenomenon. We now show that MV fusion-inhibitory peptides do not interfere with the induction of immunosuppression in vitro, indicating that MV F-H-mediated fusion is essentially not involved in this process. Proteolytic cleavage of MV F0 protein by cellular proteases, such as furin, into the F1-F2 subunits is, however, an absolute requirement, since (i) the inhibitory activity of MV-infected BJAB cells was significantly impaired in the presence of a furin-inhibitory peptide and (ii) cells expressing or viruses containing uncleaved F0 proteins revealed a strongly reduced inhibitory activity which was improved following trypsin treatment. The low inhibitory activity of effector structures containing mainly F0 proteins was not due to an impaired F0-H interaction, since both surface expression and cocapping efficiencies were similar to those found with the authentic MV F and H proteins. These results indicate that the fusogenic activity of the MV F-H complexes can be uncoupled from their immunosuppressive activity and that the immunosuppressive domains of these proteins are exposed only after proteolytic activation of the MV F0 protein. PMID:10644371

  5. Decoding distinct membrane interactions of HIV-1 fusion inhibitors using a combined atomic force and fluorescence microscopy approach.

    PubMed

    Franquelim, Henri G; Gaspar, Diana; Veiga, A Salomé; Santos, Nuno C; Castanho, Miguel A R B

    2013-08-01

    Enfuvirtide and T-1249 are two potent HIV-1 fusion inhibitor peptides. Recent studies indicate that lipids play an important role in the mode of action of those bioactive molecules. Using a combined tandem atomic force microscopy (AFM)-epifluorescence microscopy approach, we studied the interaction of both enfuvirtide and T-1249 with supported lipid bilayers. Fluid (ld)-gel (so) and ld-liquid ordered (lo) phase-separated membrane systems were tested. Results, especially for T-1249, show significant lipid membrane activity at a 15μM peptide concentration. T-1249, in opposition to enfuvirtide, induces an increase in membrane surface roughness, decrease in membrane fluidity, bilayer thinning at ld domains and disruption of the so domain borders. In terms of structural properties, both enfuvirtide and T-1249 possess distinct functional hydrophobic and amphipathic domains of HIV gp41. While enfuvirtide only yields the tryptophan-rich domain (TRD), T-1249 possesses both TRD and pocket-binding domain (PBD). TRD increases the hydrophobicity of the peptide while PBD enhances the amphipathic characteristics. As such, the enhanced membrane activity of T-1249 may be explained by a synergism between its amphipathic N-terminal segment and its hydrophophic C-terminal. Our findings provide valuable insights on the molecular-level mode of action of HIV-1 fusion inhibitors, unraveling the correlation between their structural properties and membrane interactions as a factor influencing their antiviral activity. Ultimately, this work validates the applicability of a combined AFM and fluorescence approach to evaluate the mechanic and structural properties of supported lipid bilayers upon interaction with membrane-active peptides.

  6. Vesicular PtdIns(3,4,5)P3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion.

    PubMed

    Lete, Marta G; Byrne, Richard D; Alonso, Alicia; Poccia, Dominic; Larijani, Banafshé

    2017-01-15

    Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion might also be conserved during the formation of both pronuclear and zygote nuclear envelopes in the fertilised egg. Here, we determine that class-I phosphoinositide 3-kinases (PI3Ks) are needed for in vitro nuclear envelope formation. We show that, in vivo, PtdIns(3,4,5)P3 is transiently located in vesicles around the male pronucleus at the time of nuclear envelope formation, and around male and female pronuclei before membrane fusion. We illustrate that class-I PI3K activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, using coincidence amplified Förster resonance energy transfer (FRET) monitored using fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P3 that occurs during pronuclear membrane fusion to create the zygote nuclear envelope. We present a working model, which includes several molecular steps in the pathways controlling fusion of nuclear envelope membranes.

  7. Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis

    PubMed Central

    Zucchi, Paola C.; Zick, Michael

    2011-01-01

    The fusion of sealed biological membranes joins their enclosed aqueous compartments while mixing their membrane bilayers. Reconstituted fusion reactions are commonly assayed by lipid mixing, which can result from either true fusion or from lysis and its attendant reannealing of membranes. Fusion is also frequently assayed by the mixing of lumenal aqueous compartments, using probes of low molecular weight. With several probes (biotin, methylumbelliferyl-N-acetyl-α-d-neuraminic acid, and dithionite), we find that yeast vacuolar SNAREs (SNAP [Soluble NSF attachment protein] Receptors) increase the permeability of membranes to small molecules and that this permeabilization is enhanced by homotypic fusion and vacuole protein sorting complex (HOPS) and Sec17p/Sec18p, the vacuolar tethering and SNARE chaperone proteins. We now report the development of a novel assay that allows the parallel assessment of lipid mixing, the mixing of intact lumenal compartments, any lysis that occurs, and the membrane permeation of small molecules. Applying this assay to an all-purified reconstituted system consisting of vacuolar lipids, the four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, the Rab Ypt7p, and the Rab effector/SM protein complex HOPS, we show that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and a measurable rate of lysis. PMID:21976702

  8. The IM30/Vipp1 C-terminus associates with the lipid bilayer and modulates membrane fusion.

    PubMed

    Hennig, Raoul; West, Ana; Debus, Martina; Saur, Michael; Markl, Jürgen; Sachs, Jonathan N; Schneider, Dirk

    2017-02-01

    IM30/Vipp1 proteins are crucial for thylakoid membrane biogenesis in chloroplasts and cyanobacteria. A characteristic C-terminal extension distinguishes these proteins from the homologous bacterial PspA proteins, and this extension has been discussed to be key for the IM30/Vipp1 activity. Here we report that the extension of the Synechocystis IM30 protein is indispensable, and argue that both, the N-terminal PspA-domain as well as the C-terminal extension are needed in order for the IM30 protein to conduct its in vivo function. In vitro, we show that the PspA-domain of IM30 is vital for stability/folding and oligomer formation of IM30 as well as for IM30-triggered membrane fusion. In contrast, the IM30 C-terminal domain is involved in and necessary to stabilize defined contacts to negatively charged membrane surfaces, and to modulate the IM30-induced membrane fusion activity. Although the two IM30 protein domains have distinct functional roles, only together they enable IM30 to work properly.

  9. Membrane insertion of fusion peptides from Ebola and Marburg viruses studied by replica-exchange molecular dynamics simulations.

    PubMed

    Olson, Mark A; Lee, Michael S; Yeh, In-Chul

    2017-01-28

    This work presents replica-exchange molecular dynamics simulations of inserting a 16-residue Ebola virus fusion peptide into a membrane bilayer. A computational approach is applied for modeling the peptide at the explicit all-atom level and the membrane-aqueous bilayer by a generalized Born continuum model with a smoothed switching function (GBSW). We provide an assessment of the model calculations in terms of three metrics: (1) the ability to reproduce the NMR structure of the peptide determined in the presence of SDS micelles and comparable structural data on other fusion peptides; (2) determination of the effects of the mutation Trp-8 to Ala and sequence discrimination of the homologous Marburg virus; and (3) calculation of potentials of mean force for estimating the partitioning free energy and their comparison to predictions from the Wimley-White interfacial hydrophobicity scale. We found the GBSW implicit membrane model to produce results of limited accuracy in conformational properties of the peptide when compared to the NMR structure, yet the model resolution is sufficient to determine the effect of sequence differentiation on peptide-membrane integration. © 2016 Wiley Periodicals, Inc.

  10. Characterization of Xenopus egg membrane microdomains containing uroplakin Ib/III complex: roles of their molecular interactions for subcellular localization and signal transduction.

    PubMed

    Mahbub Hasan, A K M; Ou, Zhize; Sakakibara, Keiichi; Hirahara, Shino; Iwasaki, Tetsushi; Sato, Ken-ichi; Fukami, Yasuo

    2007-02-01

    A single-transmembrane protein uroplakin III (UPIII) and its tetraspanin binding-partner uroplakin Ib (UPIb) are members of the UP proteins that were originally identified in mammalian urothelium. In Xenopus laevis eggs, these proteins: xUPIII and xUPIb, are components of the cholesterol-enriched membrane microdomains or "rafts" and involved in the sperm-egg membrane interaction and subsequent egg activation signaling via Src tyrosine kinase at fertilization. Here, we investigate whether the xUPIII-xUPIb complex is in close proximity to CD9, a tetraspanin that has been implicated in the sperm-egg fusion in the mouse and GM1, a ganglioside typically enriched in egg rafts. Preparation of the egg membrane microdomains using different non-ionic detergents (Brij 98 and Triton X-100), chemical cross-linking, co-immunoprecipitation, in vitro kinase assay and in vitro fertilization experiments demonstrated that GM1, but not CD9, is in association with the xUPIII-xUPIb complex and contributes to the sperm-dependent egg activation. Transfection experiments using HEK293 cells demonstrated that xUPIII and xUPIb localized efficiently to the cholesterol-dependent membrane microdomains when they were co-expressed, whereas co-expression of xUPIII and CD9, instead of xUPIb, did not show this effect. Furthermore, xUPIII and xUPIb were shown to suppress kinase activity of the wild type, but not a constitutively active form of, Xenopus Src protein co-expressed in HEK293 cells. These results provide novel insight into the molecular architecture of the egg membrane microdomains containing xUPIII, xUPIb and Src, which may contribute to the understanding of sperm-egg interaction and signaling during Xenopus fertilization.

  11. 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.

  12. Structural Rearrangements of the Central Region of the Morbillivirus Attachment Protein Stalk Domain Trigger F Protein Refolding for Membrane Fusion*

    PubMed Central

    Ader, Nadine; Brindley, Melinda A.; Avila, Mislay; Origgi, Francesco C.; Langedijk, Johannes P. M.; Örvell, Claes; Vandevelde, Marc; Zurbriggen, Andreas; Plemper, Richard K.; Plattet, Philippe

    2012-01-01

    It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91–115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111–118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism. PMID:22431728

  13. 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.

  14. 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.

  15. Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay.

    PubMed

    Yamamoto, Mizuki; Matsuyama, Shutoku; Li, Xiao; Takeda, Makoto; Kawaguchi, Yasushi; Inoue, Jun-Ichiro; Matsuda, Zene

    2016-11-01

    Middle East respiratory syndrome (MERS) is an emerging infectious disease associated with a relatively high mortality rate of approximately 40%. MERS is caused by MERS coronavirus (MERS-CoV) infection, and no specific drugs or vaccines are currently available to prevent MERS-CoV infection. MERS-CoV is an enveloped virus, and its envelope protein (S protein) mediates membrane fusion at the plasma membrane or endosomal membrane. Multiple proteolysis by host proteases, such as furin, transmembrane protease serine 2 (TMPRSS2), and cathepsins, causes the S protein to become fusion competent. TMPRSS2, which is localized to the plasma membrane, is a serine protease responsible for the proteolysis of S in the post-receptor-binding stage. Here, we developed a cell-based fusion assay for S in a TMPRSS2-dependent manner using cell lines expressing Renilla luciferase (RL)-based split reporter proteins. S was stably expressed in the effector cells, and the corresponding receptor for S, CD26, was stably coexpressed with TMPRSS2 in the target cells. Membrane fusion between these effector and target cells was quantitatively measured by determining the RL activity. The assay was optimized for a 384-well format, and nafamostat, a serine protease inhibitor, was identified as a potent inhibitor of S-mediated membrane fusion in a screening of about 1,000 drugs approved for use by the U.S. Food and Drug Administration. Nafamostat also blocked MERS-CoV infection in vitro Our assay has the potential to facilitate the discovery of new inhibitors of membrane fusion of MERS-CoV as well as other viruses that rely on the activity of TMPRSS2.

  16. 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%.

  17. 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.

  18. 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.

  19. 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

  20. Doc2b promotes GLUT4 exocytosis by activating the SNARE-mediated fusion reaction in a calcium- and membrane bending-dependent manner.

    PubMed

    Yu, Haijia; Rathore, Shailendra S; Davis, Eric M; Ouyang, Yan; Shen, Jingshi

    2013-04-01

    The glucose transporter GLUT4 plays a central role in maintaining body glucose homeostasis. On insulin stimulation, GLUT4-containing vesicles fuse with the plasma membrane, relocating GLUT4 from intracellular reservoirs to the cell surface to uptake excess blood glucose. The GLUT4 vesicle fusion reaction requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) as the core fusion engine and a group of regulatory proteins. In particular, the soluble C2-domain factor Doc2b plays a key role in GLUT4 vesicle fusion, but its molecular mechanism has been unclear. Here we reconstituted the SNARE-dependent GLUT4 vesicle fusion in a defined proteoliposome fusion system. We observed that Doc2b binds to GLUT4 exocytic SNAREs and potently accelerates the fusion kinetics in the presence of Ca(2+). The stimulatory activity of Doc2b requires intact Ca(2+)-binding sites on both the C2A and C2B domains. Using electron microscopy, we observed that Doc2b strongly bends the membrane bilayer, and this membrane-bending activity is essential to the stimulatory function of Doc2b in fusion. These results demonstrate that Doc2b promotes GLUT4 exocytosis by accelerating the SNARE-dependent fusion reaction by a Ca(2+)- and membrane bending-dependent mechanism. Of importance, certain features of Doc2b function appear to be distinct from how synaptotagmin-1 promotes synaptic neurotransmitter release, suggesting that exocytic Ca(2+) sensors may possess divergent mechanisms in regulating vesicle fusion.

  1. 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

  2. Extraction of lutetium(III) from aqueous solutions by employing a single fibre-supported liquid membrane.

    PubMed

    Trtić-Petrović, Tatjana M; Kumrić, Ksenija R; Dordević, Jelena S; Vladisavljević, Goran T

    2010-07-01

    Transport behaviour of Lu(III) across a polypropylene hollow fibre-supported liquid membrane containing di(2-ethylhexyl)phosphoric acid (DEHPA) in dihexyl ether as a carrier has been studied. The donor phase was LuCl(3) in the buffer solution consisting of 0.2 M sodium acetate at pH 2.5-5.0. A miniaturised system with a single hollow fibre has been operated in a batch mode. The concentration of Lu(III) was determined by indirect voltammetric method using Zn-EDTA complex. The effect of pH and volume of the donor phase, DEHPA concentration in the organic (liquid membrane) phase, the time of extraction and the content of the acceptor phase on the Lu(III) extraction and stripping behaviour was investigated. The results were discussed in terms of the pertraction and removal efficiency, the memory effect and the mean flux of Lu(III). The optimal conditions for the removal of (177)Lu(III) from labelled (177)Lu-radiopharmaceuticals were discussed and identified. The removal efficiency of Lu(III) greater than 99% was achieved at pH of the donor phase between 3.5 and 5.0 using DEHPA concentration in the organic phase of 0.47 M and the ratio of the donor to the acceptor phase of 182.

  3. The Epstein-Barr virus (EBV) glycoprotein B cytoplasmic C-terminal tail domain regulates the energy requirement for EBV-induced membrane fusion.

    PubMed

    Chen, Jia; Zhang, Xianming; Jardetzky, Theodore S; Longnecker, Richard

    2014-10-01

    The entry of enveloped viruses into host cells is preceded by membrane fusion, which in Epstein-Barr virus (EBV) is thought to be mediated by the refolding of glycoprotein B (gB) from a prefusion to a postfusion state. In our current studies, we characterized a gB C-terminal tail domain (CTD) mutant truncated at amino acid 843 (gB843). This truncation mutant is hyperfusogenic as monitored by syncytium formation and in a quantitative fusion assay and is dependent on gH/gL for fusion activity. gB843 can rescue the fusion function of other glycoprotein mutants that have null or decreased fusion activity in epithelial and B cells. In addition, gB843 requires less gp42 and gH/gL for fusion, and can function in fusion at a lower temperature than wild-type gB, indicating a lower energy requirement for fusion activation. Since a key step in fusion is the conversion of gB from a prefusion to an active postfusion state by gH/gL, gB843 may access this activated gB state more readily. Our studies indicate that the gB CTD may participate in the fusion function by maintaining gB in an inactive prefusion form prior to activation by receptor binding. Importance: Diseases resulting from Epstein-Barr virus (EBV) infection in humans range from the fairly benign disease infectious mononucleosis to life-threatening cancer. As an enveloped virus, EBV must fuse with a host cell membrane for entry and infection by using glycoproteins gH/gL, gB, and gp42. Among these glycoproteins, gB is thought to be the protein that executes fusion. To further characterize the function of the EBV gB cytoplasmic C-terminal tail domain (CTD) in fusion, we used a previously constructed CTD truncation mutant and studied its fusion activity in the context of other EBV glycoprotein mutants. From these studies, we find that the gB CTD regulates fusion by altering the energy requirements for the triggering of fusion mediated by gH/gL or gp42. Overall, our studies may lead to a better understanding of EBV fusion

  4. The HIV gp41 pocket binding domain enables C-terminal heptad repeat transition from mediating membrane fusion to immune modulation.

    PubMed

    Klug, Yoel A; Kapach, Gal; Rotem, Etai; Dubreuil, Benjamin; Shai, Yechiel

    2016-04-01

    For successful infection and propagation viruses must overcome many obstacles such as the immune system and entry into their host cells. HIV utilizes its trimeric envelope protein gp160, specifically the gp41 subunit, to enter its host cell. During this process, a gp41-central coiled coil is formed from three N- and three C-terminal heptad repeats, termed the six-helix bundle (SHB), which drives membrane fusion. Recently, T-cell suppression has been reported as an additional function for several regions of gp41 by interfering with the T-cell receptor (TCR) signalling cascade. One of these regions encompasses the conserved pocket binding domain (PBD) that is situated in the C-terminal heptad repeat (CHR) and stabilizes SHB formation. This could indicate that the PBD plays a role in T-cell suppression in addition to its role in membrane fusion. To investigate this dual function, we used two independent cell cultures coupled with biophysical techniques. The data reveal that the PBD mediates T-cell suppression by stabilizing a TCR-binding conformation in the membrane. Moreover, we show that the clinically used HIV fusion inhibitor T-20 did not show suppressive abilities, in contrast with the potent fusion inhibitor C34. In addition, by focusing on SHB conformation after its assembly, we shed light on a mechanism by which gp41's function alternates from membrane fusion facilitation to suppression of TCR activation.

  5. In Vivo Efficacy of Measles Virus Fusion Protein-Derived Peptides Is Modulated by the Properties of Self-Assembly and Membrane Residence.

    PubMed

    Figueira, T N; Palermo, L M; Veiga, A S; Huey, D; Alabi, C A; Santos, N C; Welsch, J C; Mathieu, C; Horvat, B; Niewiesk, S; Moscona, A; Castanho, M A R B; Porotto, M

    2017-01-01

    Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV hemagglutinin (H) and fusion (F) envelope glycoproteins; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad repeat (HR) regions of F can inhibit MV infection at the entry stage. In previous work, we have generated potent MV fusion inhibitors by dimerizing the F-derived peptides and conjugating them to cholesterol. We have shown that prophylactic intranasal administration of our lead fusion inhibitor efficiently protects from MV infection in vivo We show here that peptides tagged with lipophilic moieties self-assemble into nanoparticles until they reach the target cells, where they are integrated into cell membranes. The self-assembly feature enhances biodistribution and the half-life of the peptides, while integration into the target cell membrane increases fusion inhibitor potency. These factors together modulate in vivo efficacy. The results suggest a new framework for developing effective fusion inhibitory peptides.

  6. 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.

  7. 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.

  8. 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

  9. The ESCRT-III-interacting deubiquitinating enzyme AMSH3 is essential for degradation of ubiquitinated membrane proteins in Arabidopsis thaliana.

    PubMed

    Katsiarimpa, Anthi; Muñoz, Alfonso; Kalinowska, Kamila; Uemura, Tomohiro; Rojo, Enrique; Isono, Erika

    2014-04-01

    Post-translational modification by ubiquitin plays a key role in the regulation of endocytic degradation in which ubiquitinated plasma membrane cargos are transported to the vacuole for degradation dependent on the ESCRT (endosomal sorting complex required for transport) machinery. Arabidopsis AMSH3 (ASSOCIATED MOLECULE WITH THE SH3 DOMAIN OF STAM 3) is a deubiquitinating enzyme that interacts with at least two subunits of the ESCRT-III machinery, VPS2.1 and VPS24.1. amsh3 null mutation causes seedling lethality, and amsh3 null mutants show defects in multiple intracellular trafficking pathways. In this study, we further analyzed the amsh3 mutant phenotype and showed that amsh3 accumulates membrane-associated ubiquitinated proteins, supporting the indication that AMSH3 functions in ubiquitin-mediated endocytic degradation. In accordance with this, an enzymatic inactive variant of AMSH3 inhibits the AvrPtoB-dependent endocytic degradation of CERK1 (CHITIN ELICITOR RECEPTOR KINASE 1). Furthermore, we showed that the interaction of AMSH3 with ESCRT-III is important for its function in planta. Together, our data indicate the importance of AMSH3 and the AMSH3-ESCRT-III interaction for deubiquitination and degradation of ubiquitinated membrane substrates in plants.

  10. MISTIC-fusion proteins as antigens for high quality membrane protein antibodies

    PubMed Central

    Alves, Natalia Silva; Astrinidis, Susanne Adina; Eisenhardt, Nathalie; Sieverding, Cornelia; Redolfi, Josef; Lorenz, Michael; Weberruss, Marion; Moreno-Andrés, Daniel; Antonin, Wolfram

    2017-01-01

    Lack of high-quality antibodies against transmembrane proteins is a widely recognized hindrance in biomedical and cell biological research. Here we present a robust pipeline for the generation of polyclonal antibodies employing full-length membrane proteins as immunogens to overcome this “antibody bottleneck”. We express transmembrane proteins fused to a MISTIC fragment that enhances expression of eukaryotic membrane proteins in E. coli. Purified membrane proteins are used as immunogen for rabbit injection employing standard immunizing protocols. The raised antibodies against membrane proteins of the endoplasmic reticulum and the nuclear envelope, which we use as test cases, function in a wide range of applications and are superior to ones produced against soluble domains as immunogens. PMID:28148968

  11. A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

    SciTech Connect

    Esposito, Anthony M.; Cheung, Pamela; Swartz, Talia H.; Li, Hongru; Tsibane, Tshidi; Durham, Natasha D.; Basler, Christopher F.; Felsenfeld, Dan P.; Chen, Benjamin K.

    2016-03-15

    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. - Highlights: • Cre recombinase viral fusion assay screens cell-free or cell–cell entry inhibitors. • This Gag-iCre based assay is specific for the entry step of HIV replication. • Screened a library of known pharmacologic compounds for HIV fusion antagonists. • Many top hits were previously noted as HIV inhibitors, but here are classified as entry antagonists. Many top hits were previously noted as HIV inhibitors, but not as entry antagonists. • The assay is compatible with pseudotyping with HIV and heterologous viruses.

  12. 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.

  13. Solid-state NMR spectroscopy of the HIV gp41 membrane fusion protein supports intermolecular antiparallel β sheet fusion peptide structure in the final six-helix bundle state.

    PubMed

    Sackett, Kelly; Nethercott, Matthew J; Zheng, Zhaoxiong; Weliky, David P

    2014-03-06

    The HIV gp41 protein catalyzes fusion between viral and target cell membranes. Although the ~20-residue N-terminal fusion peptide (FP) region is critical for fusion, the structure of this region is not well characterized in large gp41 constructs that model the gp41 state at different times during fusion. This paper describes solid-state NMR (SSNMR) studies of FP structure in a membrane-associated construct (FP-Hairpin), which likely models the final fusion state thought to be thermostable trimers with six-helix bundle structure in the region C-terminal of the FP. The SSNMR data show that there are populations of FP-Hairpin with either α helical or β sheet FP conformation. For the β sheet population, measurements of intermolecular (13)C-(13)C proximities in the FP are consistent with a significant fraction of intermolecular antiparallel β sheet FP structure with adjacent strand crossing near L7 and F8. There appears to be negligible in-register parallel structure. These findings support assembly of membrane-associated gp41 trimers through interleaving of N-terminal FPs from different trimers. Similar SSNMR data are obtained for FP-Hairpin and a construct containing the 70 N-terminal residues of gp41 (N70), which is a model for part of the putative pre-hairpin intermediate state of gp41. FP assembly may therefore occur at an early fusion stage. On a more fundamental level, similar SSNMR data are obtained for FP-Hairpin and a construct containing the 34 N-terminal gp41 residues (FP34) and support the hypothesis that the FP is an autonomous folding domain.

  14. Aqueous extract from a Chaga medicinal mushroom, Inonotus obliquus (higher Basidiomycetes), prevents herpes simplex virus entry through inhibition of viral-induced membrane fusion.

    PubMed

    Pan, Hong-Hui; Yu, Xiong-Tao; Li, Ting; Wu, Hong-Ling; Jiao, Chun-Wei; Cai, Mian-Hua; Li, Xiang-Min; Xie, Yi-Zhen; Wang, Yi; Peng, Tao

    2013-01-01

    Chaga medicinal mushroom, Inonotus obliquus, a popular prescription in traditional medicine in Europe and Asia, was used to reduce inflammation in the nasopharynx and to facilitate breathing. The aqueous extract from I. obliquus (AEIO) exhibited marked decrease in herpes simplex virus (HSV) infection (the 50% inhibitory concentration was 3.82 μg/mL in the plaque reduction assay and 12.29 μg/mL in the HSV-1/blue assay) as well as safety in Vero cells (the 50% cellular cytotoxicity was > 1 mg/mL, and selection index was > 80). Using a time course assay, effective stage analysis, and fusion inhibition assay, the mechanism of anti-HSV activity was found against the early stage of viral infection through inhibition of viral-induced membrane fusion. Therefore, AEIO could effectively prevent HSV-1 entry by acting on viral glycoproteins, leading to the prevention of membrane fusion, which is different from nucleoside analog antiherpetics.

  15. 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

  16. 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.

  17. 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…

  18. The Type VI secretion system spike protein VgrG5 mediates membrane fusion during intercellular spread by pseudomallei group Burkholderia species.

    PubMed

    Toesca, Isabelle J; French, Christopher T; Miller, Jeff F

    2014-04-01

    Pseudomallei group Burkholderia species are facultative intracellular parasites that spread efficiently from cell to cell by a mechanism involving the fusion of adjacent cell membranes. Intercellular fusion requires the function of the cluster 5 type VI secretion system (T6SS-5) and its associated valine-glycine repeat protein, VgrG5. Here we show that VgrG5 alleles are conserved and functionally interchangeable between Burkholderia pseudomallei and its relatives B. mallei, B. oklahomensis, and B. thailandensis. We also demonstrate that the integrity of the VgrG5 C-terminal domain is required for fusogenic activity, and we identify sequence motifs, including two hydrophobic segments, that are important for fusion. Mutagenesis and secretion experiments using B. pseudomallei strains engineered to express T6SS-5 in vitro show that the VgrG5 C-terminal domain is dispensable for T6SS-mediated secretion of Hcp5, demonstrating that the ability of VgrG5 to mediate membrane fusion can be uncoupled from its essential role in type VI secretion. We propose a model in which a unique fusogenic activity at the C terminus of VgrG5 facilitates intercellular spread by B. pseudomallei and related species following injection across the plasma membranes of infected cells.

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

    PubMed Central

    Chen, Chen; Zhao, Guozhong

    2015-01-01

    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

  20. Dissociation of the trimeric gp41 ectodomain at the lipid-water interface suggests an active role in HIV-1 Env-mediated membrane fusion.

    PubMed

    Roche, Julien; Louis, John M; Grishaev, Alexander; Ying, Jinfa; Bax, Adriaan

    2014-03-04

    The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. The actual fusion process involves a switch from a homotrimeric prehairpin intermediate conformation, consisting of parallel coiled-coil helices, to a postfusion state where the ectodomains are arranged as a trimer of helical hairpins, adopting a six-helix bundle (6HB) state. Here, we show by solution NMR spectroscopy that a water-soluble 6HB gp41 ectodomain binds to zwitterionic detergents that contain phosphocholine or phosphatidylcholine head groups and phospholipid vesicles that mimic T-cell membrane composition. Binding results in the dissociation of the 6HB and the formation of a monomeric state, where its two α-helices, N-terminal heptad repeat (NHR) and C-terminal heptad repeat (CHR), become embedded in the lipid-water interface of the virus and host cell. The atomic structure of the gp41 ectodomain monomer, based on NOE distance restraints and residual dipolar couplings, shows that the NHR and CHR helices remain mostly intact, but they completely lose interhelical contacts. The high affinity of the ectodomain helices for phospholipid surfaces suggests that unzippering of the prehairpin intermediate leads to a state where the NHR and CHR helices become embedded in the host cell and viral membranes, respectively, thereby providing a physical force for bringing these membranes into close juxtaposition before actual fusion.

  1. 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.

  2. Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes

    PubMed Central

    1978-01-01

    Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'- tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++- independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259). PMID:211140

  3. Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.

    PubMed Central

    Hayashi, T; Yamasaki, S; Nauenburg, S; Binz, T; Niemann, H

    1995-01-01

    The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi et al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the N-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP. alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated. Images PMID:7774590

  4. Oviductosome-Sperm Membrane Interaction in Cargo Delivery: DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM).

    PubMed

    Al-Dossary, Amal A; Bathala, Pradeepthi; Caplan, Jeffrey L; Martin-DeLeon, Patricia A

    2015-07-17

    Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca(2+) efflux pump, plasma membrane Ca(2+)ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4-64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the αv integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving αvβ3 and α5β1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes.

  5. Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes.

    PubMed

    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.

  6. 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.

  7. A high throughput Cre–lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry

    PubMed Central

    Esposito, Anthony M.; Cheung, Pamela; Swartz, Talia H.; Li, Hongru; Tsibane, Tshidi; Durham, Natasha D.; Basler, Christopher F.; Felsenfeld, Dan P.; Chen, Benjamin K.

    2016-01-01

    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. PMID:26803470

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways

    PubMed Central

    Alto, Neal M.; Weflen, Andrew W.; Rardin, Matthew J.; Yarar, Defne; Lazar, Cheri S.; Tonikian, Raffi; Koller, Antonius; Taylor, Susan S.; Boone, Charles; Sidhu, Sachdev S.; Schmid, Sandra L.; Hecht, Gail A.; Dixon, Jack E.

    2007-01-01

    Bacterial toxins and effector proteins hijack eukaryotic enzymes that are spatially localized and display rapid signaling kinetics. However, the molecular mechanisms by which virulence factors engage highly dynamic substrates in the host cell environment are poorly understood. Here, we demonstrate that the enteropathogenic Escherichia coli (EPEC) type III effector protein EspF nucleates a multiprotein signaling complex composed of eukaryotic sorting nexin 9 (SNX9) and neuronal Wiskott-Aldrich syndrome protein (N-WASP). We demonstrate that a specific and high affinity association between EspF and SNX9 induces membrane remodeling in host cells. These membrane-remodeling events are directly coupled to N-WASP/Arp2/3–mediated actin nucleation. In addition to providing a biochemical mechanism of EspF function, we find that EspF dynamically localizes to membrane-trafficking organelles in a spatiotemporal pattern that correlates with SNX9 and N-WASP activity in living cells. Thus, our findings suggest that the EspF-dependent assembly of SNX9 and N-WASP represents a novel form of signaling mimicry used to promote EPEC pathogenesis and gastrointestinal disease. PMID:17893247

  13. 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)

  14. 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

  15. 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

    2009-01-09

    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.

  16. High performance oxygen sensing nanofibrous membranes of Eu(III) complex/polystyrene prepared by electrospinning

    NASA Astrophysics Data System (ADS)

    Yingkui, Li

    2011-07-01

    In this paper, we report the synthesis, characterization, crystal structure, and photophysical properties of a Eu 3+ complex of Eu(TTA) 3Phen, where TTA = 2-thenoyltrifluoroacetonate, and Phen = 1,10-phenanthroline. Its elementary application for oxygen-sensing application is also investigated by doping it into a polymer matrix of polystyrene (PS). Experimental data suggest that the 3 wt% doped Eu(TTA) 3Phen nanofibrous membrane exhibits a high sensitivity of 3.4 towards oxygen with a good linear relationship of R2 = 0.996. In addition, the 3 wt% doped Eu(TTA) 3Phen nanofibrous membrane owns a quick response of 9 s towards molecular oxygen, along with its excellent atmosphere insensitivity and photobleaching resistance. All these results suggest that both Eu(TTA) 3Phen and Eu(TTA) 3Phen/PS system are promising candidates for oxygen-sensing optical sensors.

  17. Receptor signaling lymphocyte-activation molecule family 1 (Slamf1) regulates membrane fusion and NADPH oxidase 2 (NOX2) activity by recruiting a Beclin-1/Vps34/ultraviolet radiation resistance-associated gene (UVRAG) complex.

    PubMed

    Ma, Chunyan; Wang, Ninghai; Detre, Cynthia; Wang, Guoxing; O'Keeffe, Michael; Terhorst, Cox

    2012-05-25

    Phagocytosis is a pivotal process by which macrophages eliminate microorganisms upon recognition by pathogen sensors. Surprisingly, the self-ligand cell surface receptor Slamf1 functions not only as a co-stimulatory molecule but also as a microbial sensor of several Gram-negative bacteria. Upon entering the phagosome of macrophages Slamf1 induces production of phosphatidylinositol 3-phosphate, which positively regulates the activity of the NOX2 enzyme and phagolysosomal maturation. Here, we report that in Escherichia coli-containing phagosomes of mouse macrophages, Slamf1 interacts with the class III PI3K Vps34 in a complex with Beclin-1 and UVRAG. Upon phagocytosis of bacteria the NOX2 activity was reduced in macrophages isolated from Beclin-1(+/-) mice compared with wild-type mice. This Slamf1/Beclin-1/Vps34/UVRAG protein complex is formed in intracellular membrane compartments as it is found without inducing phagocytosis in macrophages, human chronic lymphocytic leukemia cells, and transfectant HEK293 cells. Elimination of its cytoplasmic tail abolished the interaction of Slamf1 with the complex, but deletion or mutation of the two ITAM motifs did not. Both the BD and CCD domains of Beclin-1 were required for efficient binding to Slamf1. Because Slamf1 did not interact with Atg14L or Rubicon, which can also form a complex with Vps34 and Beclin-1, we conclude that Slamf1 recruits a subset of Vps34-associated proteins, which is involved in membrane fusion and NOX2 regulation.

  18. Truncation of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein defines elements required for fusion, incorporation, and infectivity.

    PubMed

    Yue, Ling; Shang, Liang; Hunter, Eric

    2009-11-01

    The membrane-spanning domain (MSD) of the envelope (Env) glycoprotein from human (HIV) and simian immunodeficiency viruses plays a key role in anchoring the Env complex into the viral membrane but also contributes to its biological function in fusion and virus entry. In HIV type 1 (HIV-1), it has been predicted to span 27 amino acids, from lysine residue 681 to arginine 707, and encompasses an internal arginine at residue 694. By examining a series of C-terminal-truncation mutants of the HIV-1 gp41 glycoprotein that substituted termination codons for amino acids 682 to 708, we show that this entire region is required for efficient viral infection of target cells. Truncation to the arginine at residue 694 resulted in an Env complex that was secreted from the cells. In contrast, a region from residues 681 to 698, which contains highly conserved hydrophobic residues and glycine motifs and extends 4 amino acids beyond 694R, can effectively anchor the protein in the membrane, allow efficient transport to the plasma membrane, and mediate wild-type levels of cell-cell fusion. However, these fusogenic truncated Env mutants are inefficiently incorporated into budding virions. Based on the analysis of these mutants, a "snorkeling" model, in which the flanking charged amino acid residues at 681 and 694 are buried in the lipid while their side chains interact with polar head groups, is proposed for the HIV-1 MSD.

  19. Verification of gyrokinetic particle simulation of current-driven instability in fusion plasmas. III. Collisionless tearing mode

    SciTech Connect

    Liu, Dongjian; Bao, Jian; Han, Tao; Wang, Jiaqi; Lin, Zhihong

    2016-02-15

    A finite-mass electron fluid model for low frequency electromagnetic fluctuations, particularly the collisionless tearing mode, has been implemented in the gyrokinetic toroidal code. Using this fluid model, linear properties of the collisionless tearing mode have been verified. Simulations verify that the linear growth rate of the single collisionless tearing mode is proportional to D{sub e}{sup 2}, where D{sub e} is the electron skin depth. On the other hand, the growth rate of a double tearing mode is proportional to D{sub e} in the parameter regime of fusion plasmas.

  20. Domain III substitution in Bacillus thuringiensis delta-endotoxin CryIA(b) results in superior toxicity for Spodoptera exigua and altered membrane protein recognition.

    PubMed Central

    de Maagd, R A; Kwa, M S; van der Klei, H; Yamamoto, T; Schipper, B; Vlak, J M; Stiekema, W J; Bosch, D

    1996-01-01

    To test our hypothesis that substitution of domain III of Bacillus thuringiensis delta-endotoxin (Cry) proteins might improve toxicity to pest insects, e.g., Spodoptera exigua, in vivo recombination was used to produce a number of cryIA(b)-cryIC hybrid genes. A rapid screening assay was subsequently exploited to select hybrid genes encoding soluble protoxins. Screening of 120 recombinants yielded two different hybrid genes encoding soluble proteins with domains I and II of CryIA(b) and domain III of CryIC. These proteins differed by only one amino acid residue. Both hybrid protoxins gave a protease-resistant toxin upon in vitro activation by trypsin. Bioassays showed that one of these CryIA(b)-CryIC hybrid proteins (H04) was highly toxic to S. exigua compared with the parental CryIA(b) protein and significantly more toxic than CryIC. In semiquantitative binding studies with biotin-labelled toxins and intact brush border membrane vesicles of S. exigua, this domain III substitution appeared not to affect binding-site specificity. However, binding to a 200-kDa protein by CryIA(b) in preparations of solubilized and blotted brush border membrane vesicle proteins was completely abolished by the domain III substitution. A reciprocal hybrid containing domains I and II of CryIC and domain III of CryIA(b) did bind to the 200-kDa protein, confirming that domain III of CryIA(b) was essential for this reaction. These results show that domain III of CryIC protein plays an important role in the level of toxicity to S. exigua, that substitution of domain III may be a powerful tool to increase the repertoire of available active toxins for pest insects, and that domain III is involved in binding to gut epithelium membrane proteins of S. exigua. PMID:8633853

  1. Roles of the highly conserved amino acids in the globular head and stalk region of the Newcastle disease virus HN protein in the membrane fusion process.

    PubMed

    Sun, Chengxi; Wen, Hongling; Chen, Yuzhen; Chu, Fulu; Lin, Bin; Ren, Guijie; Song, Yanyan; Wang, Zhiyu

    2015-02-01

    Newcastle disease virus (NDV), an avain paramyxovirus, has been assigned to the genus Avulavirus within the family Paramyxoviridae. It causes Newcastle disease (ND) that is a highly contagious and fatal viral disease affecting poultry and most species of birds. The hemagglutinin-neuraminidase (HN) protein of NDV has multiple functions including mediating hemadsorption (HAD), neuraminidase (NA), and fusion promotion activities affecting the process of viral attachment, entry, replication and dissemination. Fusion ability of the NDV was highly correlated to its virulence. Mutations in the HN globular head and headless HN of NDV were constructed to determinate the impact of highly conserved amino acids in the globular head of paramyxovirus HN proteins and the roles of the stalk region of HN in the fusion process. It was found that the interaction between F and HN mutants E401A, G402A, G468A, V469A, Y526A, and T527A was equal to that in F and wt HN. The mutations of G402A, G468A, V469A, and T527A had various effects on cell fusion promotion, receptor binding ability, and NA activity, but the membrane merging rate was comparable to wt HN. The elimination of hemadsorption ability and NA activity of E401A and Y526A resulted in the loss of the fusion promotion function of HN. The conclusion was that receptor binding and NA had a common active site and E401 and Y526 amino acids were essential for virus attachment, entry, and dissemination. In addition, G468A mutation made different contributions to HAD and NA, which indicated that G468 was one of the potential key amino acids in switching the two functions between receptor binding and sialic acid destruction of HN. It was also proven that the headless HN of NDV could promote the fusion event mediated by F. Thus, it revealed a novel mechanism in F activation of NDV.

  2. Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris

    PubMed Central

    Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

    2017-01-01

    Many bacterial pathogens employ the type III secretion system (T3SS) to translocate effector proteins into eukaryotic cells to overcome host defenses. To date, most of our knowledge about the T3SS molecular architecture comes from the studies on animal pathogens. In plant pathogens, nine Hrc proteins are believed to be structural components of the T3SS, of which HrcC and HrcJ form the outer and inner rings of the T3SS, respectively. Here, we demonstrated that a novel outer membrane-bound protein (HpaM) of Xanthomonas campestris pv. campestris is critical for the type III secretion and is structurally and functionally conserved in phytopathogenic Xanthomonas spp. We showed that the C-terminus of HpaM extends into the periplasm to interact physically with HrcJ and the middle part of HpaM interacts physically with HrcC. It is clear that the outer and inner rings compose the main basal body of the T3SS apparatus in animal pathogens. Therefore, we presume that HpaM may act as a T3SS structural component, or play a role in assisting assembling or affecting the stability of the T3SS apparatus. HpaM is a highly prevalent and specific protein in Xanthomonas spp., suggesting that the T3SS of Xanthomonas is distinctive in some aspects from other pathogens. PMID:28198457

  3. Identification of a novel type III secretion-associated outer membrane-bound protein from Xanthomonas campestris pv. campestris.

    PubMed

    Li, Lei; Li, Rui-Fang; Ming, Zhen-Hua; Lu, Guang-Tao; Tang, Ji-Liang

    2017-02-15

    Many bacterial pathogens employ the type III secretion system (T3SS) to translocate effector proteins into eukaryotic cells to overcome host defenses. To date, most of our knowledge about the T3SS molecular architecture comes from the studies on animal pathogens. In plant pathogens, nine Hrc proteins are believed to be structural components of the T3SS, of which HrcC and HrcJ form the outer and inner rings of the T3SS, respectively. Here, we demonstrated that a novel outer membrane-bound protein (HpaM) of Xanthomonas campestris pv. campestris is critical for the type III secretion and is structurally and functionally conserved in phytopathogenic Xanthomonas spp. We showed that the C-terminus of HpaM extends into the periplasm to interact physically with HrcJ and the middle part of HpaM interacts physically with HrcC. It is clear that the outer and inner rings compose the main basal body of the T3SS apparatus in animal pathogens. Therefore, we presume that HpaM may act as a T3SS structural component, or play a role in assisting assembling or affecting the stability of the T3SS apparatus. HpaM is a highly prevalent and specific protein in Xanthomonas spp., suggesting that the T3SS of Xanthomonas is distinctive in some aspects from other pathogens.

  4. 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.

  5. 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

    2014-01-05

    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.

  6. 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.

  7. 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

  8. Cell fusion and nuclear fusion in plants.

    PubMed

    Maruyama, Daisuke; Ohtsu, Mina; Higashiyama, Tetsuya

    2016-12-01

    Eukaryotic cells are surrounded by a plasma membrane and have a large nucleus containing the genomic DNA, which is enclosed by a nuclear envelope consisting of the outer and inner nuclear membranes. Although these membranes maintain the identity of cells, they sometimes fuse to each other, such as to produce a zygote during sexual reproduction or to give rise to other characteristically polyploid tissues. Recent studies have demonstrated that the mechanisms of plasma membrane or nuclear membrane fusion in plants are shared to some extent with those of yeasts and animals, despite the unique features of plant cells including thick cell walls and intercellular connections. Here, we summarize the key factors in the fusion of these membranes during plant reproduction, and also focus on "non-gametic cell fusion," which was thought to be rare in plant tissue, in which each cell is separated by a cell wall.

  9. The effect of chelating agent on the separation of Fe(III) and Ti(IV) from binary mixture solution by cation-exchange membrane.

    PubMed

    Kir, Esengül; Cengeloğlu, Yunus; Ersöz, Mustafa

    2005-12-15

    The competitive transport of Fe(III) and Ti(IV) ions and the effect of chelating agents on separation from binary mixture solutions through charged polysulfone cation-exchange membrane (SA3S) has been studied under Donnan dialysis conditions. The amount of chelating agent was taken as an equimolar of Fe(III) ion in the feed phase. In this process, the membrane separated two electrolyte solutions: the feed solution, initially containing metal salts (Fe, Ti), or metal salts solution, containing a chelating agent, and the other side (receiver solution) being HCl solution. An external potential field is not applied. It was observed that the chelating agents affect the metal transport; the transport of Fe(III) is decreased and the transport of Ti(IV) is increased.

  10. 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.

  11. Azo calix[4]arene based neodymium(III)-selective PVC membrane sensor.

    PubMed

    Menon, Shobhana K; Modi, Nishith R; Patel, Bhargav; Patel, Manishkumar B

    2011-02-15

    We found that the PVC membrane, containing azo calix[4]arene is a suitable ionophore, exhibited a Nernstian response for neodymium (Nd(3+)) ions (with slope of 19.8±0.2 mV decade(-1) for the triply charged ion) over a wide linear range of 4.0×10(-8) to 1.0×10(-1) mol L(-1) with a detection limit 1.0×10(-8) mol L(-1), a relatively fast response time, in the whole concentration range (<10 s), and a considerable life time at least for four months in the pH range of 4.0-8.0. Furthermore, the electrode revealed high selectivity with respect to all the common alkali, alkaline earth, transition and heavy metal ions, including the members of the lanthanide family other than Nd(3+). Concerning its applications, it was effectively employed for the determination of neodymium ions in industrial waste water as well as in lake water.

  12. 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

    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.

  13. 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

  14. Components of the plasma membrane of growing axons. III. Saxitoxin binding to sodium channels

    PubMed Central

    1984-01-01

    The density of sodium channels was measured in growing and mature axons of the olfactory nerve of the bullfrog, using as a probe the drug saxitoxin (STX). The toxin binds to control nerves from adult animals in a saturable manner with a dissociation constant of approximately 23 nM at 4 degrees C and a capacity of 72 fmol/mg wet weight, equivalent to about five sites per square micrometer of axolemma. In growing nerves, obtained from adult frogs 4-5 wk following removal of the original nerve, the STX-binding capacity per wet weight of tissue is markedly reduced, to approximately 25% of control values, and appears to decrease in the proximodistal direction. STX-binding data, expressed as STX/mg wet weight, was converted to STX/micron 2 of axolemma using stereologically derived values of membrane area per milligram wet weight of nerve. The axolemmal content (area/mg wet weight) of all regions of growing nerve is substantially decreased compared to controls, but increases in the proximodistal direction by 60%. These changes in axolemmal area result in calculated STX receptor densities (per unit axolemmal area) which, in distal regions, are approximately at the level of the mature nerve and, in proximal regions, are actually increased above controls by 50 to 70%. Upon comparing the axolemmal density of intramembrane particles, reported in the companion paper, with the calculated density of STX receptors in both mature and growing nerves, we find a correlation between STX receptors and intramembrane particles with diameters of 11.5-14.0 nm. The growing axon's gradient of sodium channels and the shift from this gradient to a uniform distribution in the mature axon suggest (a) that sodium channels are inserted into the perikaryal plasmalemma and diffuse from there into the growing axolemma, and (b) that the axolemma undergoes functional maturation during growth. PMID:6325471

  15. 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.

  16. Simultaneous Au(III) Extraction and In Situ Formation of Polymeric Membrane-Supported Au Nanoparticles: A Sustainable Process with Application in Catalysis.

    PubMed

    Mora-Tamez, Lucía; Esquivel-Peña, Vicente; Ocampo, Ana L; Rodríguez de San Miguel, Eduardo; Grande, Daniel; de Gyves, Josefina

    2017-01-07

    A polymeric membrane-supported catalyst with immobilized gold nanoparticles (AuNPs) was prepared through the extraction and in situ reduction of Au(III) salts in a one-step strategy. Polymeric inclusion membranes (PIMs) and polymeric nanoporous membranes (PNMs) were tested as different membrane-support systems. Transport experiments indicated that PIMs composed of cellulose triacetate, 2-nitrophenyloctyl ether, and an aliphatic tertiary amine (Adogen 364 or Alamine 336) were the most efficient supports for Au(III) extraction. The simultaneous extraction and reduction processes were proven to be the result of a synergic phenomenon in which all the membrane components were involved. Scanning electron microscopy characterization of cross-sectional samples suggested a distribution of AuNPs throughout the membrane. Transmission electron microscopy characterization of the AuNPs indicated average particle sizes of 36.7 and 2.9 nm for the PIMs and PNMs, respectively. AuNPs supported on PIMs allowed for >95.4 % reduction of a 0.05 mmol L(-1) 4-nitrophenol aqueous solution with 10 mmol L(-1) NaBH4 solution within 25 min.

  17. Observation of indirect-drive inertial confinement fusion implosion asymmetry on the Shenguang III prototype laser facility

    NASA Astrophysics Data System (ADS)

    Li, Hang

    2016-10-01

    Three kinds of hohlraum lengths were used to study the symmetry scaling on the Shenguang III prototype laser facility. Hot spot radiography was taken by an x-ray framing camera and the hot spot ellipticity a/b which showd a ``P2 like'' implosion distortion was measured. The indirect-drive implosion asymmetry is determined by the hohlraum radiation uniformity. Most factors affecting hohlraum radiation uniformity can be taken into account by a view-factor code IRAD 3D, so time-resolved difference between polar and equatorial radiation flux can by calculated by IRAD 3D. Then, the time-resolved a/b evolution can be calculated by a simplified analytic model integrating the total difference between polar and equatorial radiation flux before each moment, because during the acceleration phase the capsule distortion at some time is the accumulation effect of total radiation drive before that time. The calculated results of the time-resolved implosion asymmetry are basically in agreement with experimental results. Meanwhile, the physical mechanism for how hohlraum radiation nonuniformity evolution induces the variations of implosion asymmetry with hohlraum length and time is analyzed.

  18. 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.

  19. 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.

  20. Crystal structure of the hemolytic lectin CEL-III isolated from the marine invertebrate Cucumaria echinata: implications of domain structure for its membrane pore-formation mechanism.

    PubMed

    Uchida, Tatsuya; Yamasaki, Takayuki; Eto, Seiichiro; Sugawara, Hajime; Kurisu, Genji; Nakagawa, Atsushi; Kusunoki, Masami; Hatakeyama, Tomomitsu

    2004-08-27

    CEL-III is a Ca(2+)-dependent and galactose-specific lectin purified from the sea cucumber, Cucumaria echinata, which exhibits hemolytic and hemagglutinating activities. Six molecules of CEL-III are assumed to oligomerize to form an ion-permeable pore in the cell membrane. We have determined the crystal structure of CELIII by using single isomorphous replacement aided by anomalous scattering in lead at 1.7 A resolution. CEL-III consists of three distinct domains as follows: the N-terminal two carbohydrate-binding domains (1 and 2), which adopt beta-trefoil folds such as the B-chain of ricin and are members of the (QXW)(3) motif family; and domain 3, which is a novel fold composed of two alpha-helices and one beta-sandwich. CEL-III is the first Ca(2+)-dependent lectin structure with two beta-trefoil folds. Despite sharing the structure of the B-chain of ricin, CEL-III binds five Ca(2+) ions at five of the six subdomains in both domains 1 and 2. Considering the relatively high similarity among the five subdomains, they are putative binding sites for galactose-related carbohydrates, although it remains to be elucidated whether bound Ca(2+) is directly involved in interaction with carbohydrates. The paucity of hydrophobic interactions in the interfaces between the domains and biochemical data suggest that these domains rearrange upon carbohydrate binding in the erythrocyte membrane. This conformational change may be responsible for oligomerization of CEL-III molecules and hemolysis in the erythrocyte membranes.

  1. Specific detection of Xanthomonas oryzae pv. oryzicola in infected rice plant by use of PCR assay targeting a membrane fusion protein gene.

    PubMed

    Kang, Man Jung; Shim, Jae Kyung; Cho, Min Seok; Seol, Young Joo; Hahn, Jang Ho; Hwang, Duk Ju; Park, Dong Suk

    2008-09-01

    Successful control of Xanthomonas oryzae pv. oryzicola, the causal agent of bacterial leaf streak, requires a specific and reliable diagnostic tool. A pathovar-specific PCR assay was developed for the rapid and accurate detection of the plant pathogenic bacterium Xanthomonas oryzae pv. oryzicola in diseased plant. Based on differences in a membrane fusion protein gene of Xanthomonas oryzae pv. oryzicola and other microorganisms, which was generated from NCBI (http://www.ncbi.nlm.nih.gov/) and CMR (http://cmr.tigr.org/) BLAST searches, one pair of pathovar-specific primers, XOCMF/XOCMR, was synthesized. Primers XOCMF and XOCMR from a membrane fusion protein gene were used to amplify a 488-bp DNA fragment. The PCR product was only produced from 4 isolates of Xanthomonas oryzae pv. oryzicola among 37 isolates of other pathovars and species of Xanthomonas, Pectobacterium, Pseudomonas, Burkholderia, Escherichia coli, and Fusarium oxysporum f.sp. dianthi. The results suggested that the assay detected the pathogen more rapidly and accurately than standard isolation methods.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N.

    PubMed

    de Maagd, R A; Bakker, P L; Masson, L; Adang, M J; Sangadala, S; Stiekema, W; Bosch, D

    1999-01-01

    Three types of binding assays were used to study the binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to brush border membrane vesicle (BBMV) membranes and a purified putative receptor of the target insect Manduca sexta. Using hybrid proteins consisting of Cry1Ac and the related Cry1C protein, it was shown that domain III of Cry1Ac is involved in specificity of binding as observed by all three techniques. In ligand blotting experiments using SDS-PAGE-separated BBMV proteins as well as the purified putative receptor aminopeptidase N (APN), the presence of domain III of Cry1Ac in a hybrid with Cry1C was necessary and sufficient for specific binding to APN. Using the surface plasmon resonance (SPR) technique with immobilized APN, it was shown that the presence of domain III of Cry1Ac in a hybrid is sufficient for binding to one of the two previously identified Cry1Ac binding sites, whereas the second site requires the full Cry1Ac toxin for binding. In addition, the role of domain III in the very specific inhibition of Cry1Ac binding by the amino sugar N-acetylgalactosamine (GalNac) was determined. Both in ligand blotting and in surface plasmon resonance experiments, as well as in binding assays using intact BBMVs, it was shown that the presence of domain III of Cry1Ac in a toxin molecule is sufficient for the inhibition of binding by GalNAc. These and other results strongly suggest that domain III of delta-endotoxins play a role in insect specificity through their involvement in specific binding to insect gut epithelial receptors.

  7. End-products diacylglycerol and ceramide modulate membrane fusion induced by a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

    PubMed Central

    Ibarguren, Maitane; Bomans, Paul H. H.; Frederik, Peter M.; Stonehouse, Martin; Vasil, Michael L.; Alonso, Alicia; Goñi, Félix M.

    2009-01-01

    A phospholipase C/ sphingomyelinase from Pseudomonas aeruginosa has been assayed on vesicles containing phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and cholesterol, at equimolar ratios. The enzyme activity modifies the bilayer chemical composition giving rise to diacylglycerol (DAG) and ceramide (Cer). Assays of enzyme activity, enzyme-induced aggregation and fusion have been performed. Ultrastructural evidence of vesicle fusion at various stages of the process is presented, based on cryo-EM observations. The two enzyme lipidic end-products, DAG and Cer, have opposite effects on the bilayer physical properties, the former abolishes lateral phase separation, while the latter generates a new gel phase [Sot et al., FEBS Lett. 582, 3230–3236 (2008)]. Addition of either DAG, or Cer, or both to the liposome mixture causes an increase in enzyme binding to the bilayers and a decrease in lag time of hydrolysis. These two lipids also have different effects on the enzyme activity, DAG enhancing enzyme-induced vesicle aggregation and fusion, Cer inhibiting the hydrolytic activity. These effects are explained in terms of the different physical properties of the two lipids. DAG increases bilayers fluidity and decreases lateral separation of lipids, thus increasing enzyme activity and substrate accessibility to the enzyme. Cer has the opposite effect mainly because of its tendency to sequester sphingomyelin, an enzyme substrate, into rigid domains, presumably less accessible to the enzyme. PMID:19891956

  8. Subunit III of the chloroplast ATP-synthase can form a Ca(2+)-binding site on the lumenal side of the thylakoid membrane.

    PubMed

    Zakharov, S D; Ewy, R G; Dilley, R A

    1993-12-20

    Subunit III, the 8 kDa component of the chloroplast CFo H+ channel, was isolated and purified from pea thylakoids for the purpose of studying its Ca(2+)-binding properties. After n-butanol extraction and ether precipitation, HPLC purification was accomplished using a poly(styrene-divinylbenzene) column which removes lipid and protein contaminations. The main components of protein contamination were two hydrophobic proteins of near 4 kDa molecular mass, the psaI and psbK gene products associated with PSI and PSII reaction centers, respectively. Purified subunit III as well as the unfractionated organic-solvent soluble preparation were used in a 45Ca(2+)-ligand blot assay known to detect high affinity Ca(2+)-binding sites in proteins. Polypeptides were separated with SDS-PAGE and were transferred onto PVDF membranes. Treatment of the membrane with 45CaCl2 in the presence of 10-fold excess of MgCl2 and 200-fold excess KCl led to the labeling of only the 8 kDa polypeptide. The Ca2+ binding was inhibited after derivatizing aqueously exposed carboxyl groups with a water soluble carbodiimide plus a nucleophile, after de-formylation of the N-terminal methionine, or with a subsequent treatment with La3+. Ca2+ binding was maximum at pH 7.5-8.5 and was greatly decreased at acidic pH. Dicyclohexylcarbodiimide treatment (no nucleophile was added) of thylakoid membranes, which derivatizes the hydrophobically located Glu-61, decreased the electrophoretical mobility of isolated subunit III but did not inhibit the Ca2+ binding. The data indicate that the carbonyl group of the formylated N-terminal Met-1 and probably the carboxyl group of the subunit III C-terminal Val-81 provide some of seven essential oxygen ligands normally required for defining a Ca(2+)-binding site in proteins. It is probable, but not yet established that an oligomeric form of subunit III polypeptides is essential for forming the Ca(2+)-binding site. Based on the accepted models for the hairpin conformation of

  9. Antibody raised against soluble CD4-rgp120 complex recognizes the CD4 moiety and blocks membrane fusion without inhibiting CD4-gp120 binding

    PubMed Central

    1990-01-01

    We studied the humoral response of mice immunized with soluble CD4- rgp120 complex, testing polyclonal and monoclonal antibodies (mAbs) with the aim of identifying molecular changes that take place after the first interaction between human immunodeficiency virus and the cell surface. The antisera had a paradoxically high syncytia-blocking titer associated with anti-CD4 specificity, while their capacity to inhibit CD4-gp120 binding was relatively modest. One of the mAbs produced from these responders blocks syncytia formation but does not inhibit CD4 interaction with gp120. Apparently, this mAb interacts with the CD4 moiety of CD4-gp120 complex and prevents a post-binding event necessary for membrane fusion and viral infection. PMID:2212945

  10. ADS-J1 inhibits HIV-1 infection and membrane fusion by targeting the highly conserved pocket in the gp41 NHR-trimer.

    PubMed

    Yu, Fei; Lu, Lu; Liu, Qi; Yu, Xiaowen; Wang, Lili; He, Elaine; Zou, Peng; Du, Lanying; Sanders, Rogier W; Liu, Shuwen; Jiang, Shibo

    2014-05-01

    We previously identified a potent small-molecule human immunodeficiency virus type 1 (HIV-1) fusion inhibitor, termed ADS-J1, and hypothesized that it mainly targeted the hydrophobic pocket in the gp41 N-terminal heptad repeat (NHR) trimer. However, this hypothesis has been challenged by the fact that ADS-J1 cannot induce drug-resistance mutation in the gp41 pocket region. Therefore, we show herein that HIV-1 mutants resistant to T2635, a peptide derived from the gp41 C-terminal heptad repeat (CHR) region with pocket-binding domain (PBD), were also resistant to ADS-J1. We also show that pseudoviruses with mutations at positions 64 and 67 in the gp41 pocket region were highly resistant to ADS-J1 and C34, another CHR-peptide with PBD, but relatively sensitive to T20, a CHR-peptide without PBD. ADS-J1 could effectively bind to N36Fd, a mimic of the gp41 NHR-trimer with pocket exposed, and block binding of C34 to N36Fd trimer to form six-helix bundle (6-HB). However, ADS-J1 was less effective in binding to N36Fd trimer with mutations in the gp41 pocket region, such as N36(Q64A)Fd, N36(Q64L)Fd, N36(A67G)Fd, N36(A67S)Fd, and N36(Q66R)Fd, as well as less effective in blocking 6-HB formation between C34 and these mutant N36Fd trimers. These results confirm that ADS-J1 mainly targets the pocket region in the HIV-1 gp41 NHR trimer and suggest that it could be used as a lead for developing small-molecule HIV fusion inhibitors and as a molecule probe for studying the mechanisms of gp41-mediated membrane fusion.

  11. Autographa californica multicapsid nucleopolyhedrovirus efficiently infects Sf9 cells and transduces mammalian cells via direct fusion with the plasma membrane at low pH.

    PubMed

    Dong, Sicong; Wang, Manli; Qiu, Zhijuan; Deng, Fei; Vlak, Just M; Hu, Zhihong; Wang, Hualin

    2010-05-01

    The budded virus (BV) of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) infects insect cells and transduces mammalian cells mainly through the endocytosis pathway. However, this study revealed that the treatment of the virus bound to Sf9 cells at low pH could efficiently rescue the infectivity of AcMNPV in the presence of endocytosis pathway inhibitors. A colocalization assay of the major capsid protein VP39 with the early endosome marker EEA1 showed that at low pH, AcMNPV entered Sf9 cells via an endosome-independent pathway. Using a fluorescent probe (R18), we showed that at low pH, the viral nucleocapsid entered Sf9 cells via direct fusion at the cell surface. By using the myosin-specific inhibitor 2,3-butanedione monoxime (BDM) and the microtubule inhibitor nocodazole, the low pH-triggered direct fusion was demonstrated to be dependent on myosin-like proteins and independent of microtubules. The reverse transcription-PCR of the IE1 gene as a marker for viral entry showed that the kinetics of AcMNPV in cells triggered by low pH was similar to that of the normal entry via endocytosis. The low pH-mediated infection assay and VP39 and EEA1 colocalization assay also demonstrated that AcMNPV could efficiently transduce mammalian cells via direct membrane fusion at the cell surface. More importantly, we found that a low-pH trigger could significantly improve the transduction efficiency of AcMNPV in mammalian cells, leading to the potential application of this method when using baculovirus as a vector for heterologous gene expression and for gene therapy.

  12. 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.

  13. Store-activated Ca2+ inflow in Xenopus laevis oocytes: inhibition by primaquine and evaluation of the role of membrane fusion.

    PubMed Central

    Gregory, R B; Barritt, G J

    1996-01-01

    The role of membrane fusion in the activation of store-activated Ca2+ channels (SACCs) in the plasma membrane of Xenopus laevis oocytes was investigated with primaquine, an inhibitor of vesicle trafficking, reagents that disrupt the cytoskeleton, and reagents that activate or inhibit the functions of monomeric and trimeric GTP-binding regulatory proteins. Ca2+ inflow was assessed by measuring the rate of increase in the fluorescence of the intracellular Ca2+ chelator fluo-3 after the addition of extracellular Ca2+ to oocytes previously incubated in the absence of added Ca2+. Primaquine inhibited the 3-deoxy-3-fluoro Ins(1,4,5)P3 (Ins(1,4,5)P3F)-stimulated increase in Ca2+O,-induced fluo-3 fluorescence with no detectable effect on the release of Ca2+ from intracellular stores. The effect of primaquine was observed within 1.5 min, showed similarity to the inhibition induced by Gd3+, was reversible, and was observed when primaquine was added either before or after activation of the SACCs. The degree of inhibition of Ca2+ inflow by primaquine was halved when the extracellular concentration of Ca2+ was increased from 3.1 to 12.5 mM. Primaquine also inhibited Ca2+ inflow through cholera toxin-activated divalent cation channels and Drosophila Trpl channels (expressed in oocytes after injection of trp1 cRNA). These results indicate that primaquine inhibits open SACCs, possibly by directly inhibiting Ca2+ flow through the channel pore. Colchicine plus cytochalasin B, Brefeldin A, the peptide Arf-1 (2-17) (introduced by microinjection), lovastatin or pertussis toxin did not inhibit the Ins(1,4,5)P3F stimulated increase in fluo-3 fluorescence. In contrast, guanosine 5'-[gamma-thio]triphosphate (GTP[S]), guanosine 5'-[beta, gamma-imido]triphosphate (p[NH]ppG) and A1F4-, but not guanosine 5'-[beta-thio]diphosphate, inhibited the Ins(1,4,5)P3F-stimulated increase in fluo-3 fluorescence. Co-administration of GTP did not prevent the inhibition by GTP[S] of FA1F4-. Staurosporine

  14. 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

  15. 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

    2013-10-03

    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

  16. An aluminum selective electrode via modification of PVC membrane by modified clinoptilolite nanoparticles with hexadecyltrimethyl ammonium bromide (HDTMA-Br) surfactant containing Arsenazo III.

    PubMed

    Mahdavi, Mostafa; Nezamzadeh-Ejhieh, Alireza

    2017-05-15

    A modified PVC (polyvinyl chloride) membrane with clinoptilolite nanoparticles/hexadecyltrimethyl ammonium bromide surfactant (HDTMABr)/Arsenazo III: (NSMZ-ARS), was used for construction of Al(III) -selective electrode. The raw and modified samples were characterized by XRD, FTIR, SEM and TEM methods. Among the different tested membranes constructed by raw and modified samples, only, the membrane electrode containing NSMZ-ARS showed a suitable Nernstian response to aluminum in the concentration range of 2.04×10(-5) to 1.99×10(-1)M (r=0.9987), with a detection limit of 1.0×10(-5)molL(-1) and a Nernstian slope of 19.6±0.5mV per decade of aluminum concentration. The electrode response to aluminum remained constant in the pH range of 2-4. The sensor was selective for aluminum over a wide variety of other ions and exhibited. The electrode had a rapid response time and its response reached to steady potential at 5s. The electrode had also satisfactory long term stability and held its activity at least 3months.

  17. 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

  18. A myelin proteolipid protein-LacZ fusion protein is developmentally regulated and targeted to the myelin membrane in transgenic mice

    PubMed Central

    1993-01-01

    Transgenic mice were generated with a fusion gene carrying a portion of the murine myelin proteolipid protein (PLP) gene, including the first intron, fused to the E. coli LacZ gene. Three transgenic lines were derived and all lines expressed the transgene in central nervous system white matter as measured by a histochemical assay for the detection of beta-galactosidase activity. PLP-LacZ transgene expression was regulated in both a spatial and temporal manner, consistent with endogenous PLP expression. Moreover, the transgene was expressed specifically in oligodendrocytes from primary mixed glial cultures prepared from transgenic mouse brains and appeared to be developmentally regulated in vitro as well. Transgene expression occurred in embryos, presumably in pre- or nonmyelinating cells, rather extensively throughout the peripheral nervous system and within very discrete regions of the central nervous system. Surprisingly, beta- galactosidase activity was localized predominantly in the myelin in these transgenic animals, suggesting that the NH2-terminal 13 amino acids of PLP, which were present in the PLP-LacZ gene product, were sufficient to target the protein to the myelin membrane. Thus, the first half of the PLP gene contains sequences sufficient to direct both spatial and temporal gene regulation and to encode amino acids important in targeting the protein to the myelin membrane. PMID:8408224

  19. Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

    SciTech Connect

    Abdali, Narges; Parks, Jerry M.; Haynes, Keith M.; Chaney, Julie L.; Green, Adam T.; Wolloscheck, David; Walker, John K.; Rybenkov, Valentin V.; Baudry, Jerome; Smith, Jeremy C.; Zgurskaya, Helen I.

    2016-10-21

    Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for developing effective EPIs, especially in light of constantly emerging resistance. We describe new EPIs that interact with and possibly inhibit the function of periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump, change its structure in vivo, inhibit efflux of fluorescent probes and potentiate the activities of antibiotics in Escherichia coli cells. These findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.

  20. Reviving Antibiotics: Efflux Pump Inhibitors That Interact with AcrA, a Membrane Fusion Protein of the AcrAB-TolC Multidrug Efflux Pump

    DOE PAGES

    Abdali, Narges; Parks, Jerry M.; Haynes, Keith M.; ...

    2016-10-21

    Antibiotic resistance is a major threat to human welfare. Inhibitors of multidrug efflux pumps (EPIs) are promising alternative therapeutics that could revive activities of antibiotics and reduce bacterial virulence. Identification of new druggable sites for inhibition is critical for developing effective EPIs, especially in light of constantly emerging resistance. We describe new EPIs that interact with and possibly inhibit the function of periplasmic membrane fusion proteins, critical components of efflux pumps that are responsible for the activation of the transporter and the recruitment of the outer-membrane channel. The discovered EPIs bind to AcrA, a component of the prototypical AcrAB-TolC pump,more » change its structure in vivo, inhibit efflux of fluorescent probes and potentiate the activities of antibiotics in Escherichia coli cells. These findings expand the chemical and mechanistic diversity of EPIs, suggest the mechanism for regulation of the efflux pump assembly and activity, and provide a promising path for reviving the activities of antibiotics in resistant bacteria.« less

  1. Myristoylated and non-myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein.

    PubMed Central

    Hanakam, F; Albrecht, R; Eckerskorn, C; Matzner, M; Gerisch, G

    1996-01-01

    Hisactophilins are myristoylated proteins that are rich in histidine residues and known to exist in Dictyostelium cells in a plasma membrane-bound and a soluble cytoplasmic state. Intracellular translocation of these proteins in response to pH changes was monitored using hisactophilin fusions with green fluorescent protein (GFP) and confocal laser scanning microscopy. Both the normal and a mutated non-myristoylated fusion protein shuffled within the cells in a pH-dependent manner. After lowering the pH, these proteins translocated within minutes between the cytoplasm, the plasma membrane and the nucleus. The role of histidine clusters on the surface of hisactophilin molecules in binding of the proteins to the plasma membrane and in their transfer to the nucleus is discussed on the basis of a pH switch mechanism. Images PMID:8670794

  2. A Plasma Membrane-Anchored Fluorescent Protein Fusion Illuminates Sieve Element Plasma Membranes in Arabidopsis and Tobacco1[W][OA

    PubMed Central

    Thompson, Matthew V.; Wolniak, Stephen M.

    2008-01-01

    Rapid acquisition of quantitative anatomical data from the sieve tubes of angiosperm phloem has been confounded by their small size, their distance from organ surfaces, and the time-consuming nature of traditional methods, such as transmission electron microscopy. To improve access to these cells, for which good anatomical data are critical, a monomeric yellow fluorescent protein (mCitrine) was N-terminally fused to a small (approximately 6 kD) membrane protein (AtRCI2A) and stably expressed in Arabidopsis thaliana (Columbia-0 ecotype) and Nicotiana tabacum (‘Samsun’) under the control of a companion cell-specific promoter (AtSUC2p). The construct, called by its abbreviation SUmCR, yielded stable sieve element (SE) plasma membrane fluorescence labeling, even after plastic (methacrylate) embedding. In conjunction with wide-field fluorescence measurements of sieve pore number and position using aniline blue-stained callose, mCitrine-labeled material was used to calculate rough estimates of sieve tube-specific conductivity for both species. The SUmCR construct also revealed a hitherto unknown expression domain of the AtSUC2 Suc-H+ symporter in the epidermis of the cell division zone of developing root tips. The success of this construct in targeting plasma membrane-anchored fluorescent proteins to SEs could be attributable to the small size of AtRCI2A or to the presence of other signals innate to AtRCI2A that permit the protein to be trafficked to SEs. The construct provides a hitherto unique entrée into companion cell-to-SE protein targeting, as well as a new tool for studying whole-plant phloem anatomy and architecture. PMID:18223149

  3. Synthesis and Evaluation of GdIII-Based Magnetic Resonance Contrast Agents for Molecular Imaging of Prostate-Specific Membrane Antigen**

    PubMed Central

    Ngen, Ethel J.; Rotz, Matthew W.; Kakkad, Samata; Lisok, Ala; Pracitto, Richard; Pullambhatla, Mrudula; Chen, Zhengping; Shah, Tariq; Artemov, Dmitri; Meade, Thomas J.; Bhujwalla, Zaver M.; Pomper, Martin G.

    2016-01-01

    Magnetic resonance (MR) imaging is advantageous because it concurrently provides anatomic, functional, and molecular information. MR molecular imaging can combine the high spatial resolution of this established clinical modality with molecular profiling in vivo. However, as a result of the intrinsically low sensitivity of MR imaging, high local concentrations of biological targets are required to generate discernable MR contrast. We hypothesize that the prostate-specific membrane antigen (PSMA), an attractive target for imaging and therapy of prostate cancer, could serve as a suitable biomarker for MR-based molecular imaging. We have synthesized three new high-affinity, low-molecular-weight GdIII-based PSMA-targeted contrast agents containing one to three GdIII chelates per molecule. We evaluated the relaxometric properties of these agents in solution, in prostate cancer cells, and in an in vivo experimental model to demonstrate the feasibility of PSMA-based MR molecular imaging. PMID:26212031

  4. 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

  5. Activation of Membrane Fusion by Murine Leukemia Viruses Is Controlled in cis or in trans by Interactions between the Receptor-Binding Domain and a Conserved Disulfide Loop of the Carboxy Terminus of the Surface Glycoprotein

    PubMed Central

    Lavillette, Dimitri; Boson, Bertrand; Russell, Stephen J.; Cosset, François-Loïc

    2001-01-01

    Cell entry of retroviruses is initiated by the recognition of cellular receptors and the subsequent membrane fusion between viral and cellular membranes. These two steps are mediated by the surface (SU) and transmembrane (TM) subunits of the retroviral envelope glycoprotein (Env), respectively. Determinants regulating membrane fusion have been described throughout SU and TM, but the processes coupling receptor recognition to fusion are still elusive. Here we establish that a critical interaction is formed between the receptor-binding domain (RBD) and the major disulfide loop of the carboxy-terminal domain (C domain) of the murine leukemia virus SU. Receptor binding causes an alteration of this interaction and, in turn, promotes further events of Env fusion activation. We characterize mutations which, by lowering this interaction and reducing the compatibility between the RBD and C domains of Env glycoprotein chimeras, affect both Env fusogenicity and sensitivity to receptor interference. Additionally, we demonstrate that suboptimal interactions in such mutant Env proteins can be compensated in trans by soluble RBDs in a manner that depends on their compatibility with the C domain. Our results therefore indicate that RBD/C domain interactions may occur in cis, via the proper RBD of the viral Env itself, or in trans, via a distinct RBD expressed by virion-free Env glycoproteins expressed endogenously by the infected cells or provided by neighboring Env trimers. PMID:11264358

  6. Dynamics of rat entorhinal cortex layer II and III cells: characteristics of membrane potential resonance at rest predict oscillation properties near threshold

    PubMed Central

    Erchova, I; Kreck, G; Heinemann, U; Herz, A V M

    2004-01-01

    Neurones generate intrinsic subthreshold membrane potential oscillations (MPOs) under various physiological and behavioural conditions. These oscillations influence neural responses and coding properties on many levels. On the single-cell level, MPOs modulate the temporal precision of action potentials; they also have a pronounced impact on large-scale cortical activity. Recent studies have described a close association between the MPOs of a given neurone and its electrical resonance properties. Using intracellular sharp microelectrode recordings we examine both dynamical characteristics in layers II and III of the entorhinal cortex (EC). Our data from EC layer II stellate cells show strong membrane potential resonances and oscillations, both in the range of 5–15 Hz. At the resonance maximum, the membrane impedance can be more than twice as large as the input resistance. In EC layer III cells, MPOs could not be elicited, and frequency-resolved impedances decay monotonically with increasing frequency or has only a small peak followed by a subsequent decay. To quantify and compare the resonance and oscillation properties, we use a simple mathematical model that includes stochastic components to capture channel noise. Based on this model we demonstrate that electrical resonance is closely related though not equivalent to the occurrence of sag-potentials and MPOs. MPO frequencies can be predicted from the membrane impedance curve for stellate cells. The model also explains the broad-band nature of the observed MPOs. This underscores the importance of intrinsic noise sources for subthreshold phenomena and rules out a deterministic description of MPOs. In addition, our results show that the two identified cell classes in the superficial EC layers, which are known to target different areas in the hippocampus, also have different preferred frequency ranges and dynamic characteristics. Intrinsic cell properties may thus play a major role for the frequency

  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. 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.

  9. 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.

  10. 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.

  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. Membrane curvature at a glance

    PubMed Central

    McMahon, Harvey T.; Boucrot, Emmanuel

    2015-01-01

    ABSTRACT Membrane curvature is an important parameter in defining the morphology of cells, organelles and local membrane subdomains. Transport intermediates have simpler shapes, being either spheres or tubules. The generation and maintenance of curvature is of central importance for maintaining trafficking and cellular functions. It is possible that local shapes in complex membranes could help to define local subregions. In this Cell Science at a Glance article and accompanying poster, we summarize how generating, sensing and maintaining high local membrane curvature is an active process that is mediated and controlled by specialized proteins using general mechanisms: (i) changes in lipid composition and asymmetry, (ii) partitioning of shaped transmembrane domains of integral membrane proteins or protein or domain crowding, (iii) reversible insertion of hydrophobic protein motifs, (iv) nanoscopic scaffolding by oligomerized hydrophilic protein domains and, finally, (v) macroscopic scaffolding by the cytoskeleton with forces generated by polymerization and by molecular motors. We also summarize some of the discoveries about the functions of membrane curvature, where in addition to providing cell or organelle shape, local curvature can affect processes like membrane scission and fusion as well as protein concentration and enzyme activation on membranes. PMID:25774051

  13. 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.

  14. Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene.

    PubMed Central

    Vialard, J; Lalumière, M; Vernet, T; Briedis, D; Alkhatib, G; Henning, D; Levin, D; Richardson, C

    1990-01-01

    An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified

  15. Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion.

    PubMed

    Yang, Sung-Tae; Kiessling, Volker; Tamm, Lukas K

    2016-04-26

    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.

  16. 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.

  17. 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.

  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.

  19. Enhanced growth of influenza vaccine seed viruses in vero cells mediated by broadening the optimal pH range for virus membrane fusion.

    PubMed

    Murakami, Shin; Horimoto, Taisuke; Ito, Mutsumi; Takano, Ryo; Katsura, Hiroaki; Shimojima, Masayuki; Kawaoka, Yoshihiro

    2012-02-01

    Vaccination is one of the most effective preventive measures to combat influenza. Prospectively, cell culture-based influenza vaccines play an important role for robust vaccine production in both normal settings and urgent situations, such as during the 2009 pandemic. African green monkey Vero cells are recommended by the World Health Organization as a safe substrate for influenza vaccine production for human use. However, the growth of influenza vaccine seed viruses is occasionally suboptimal in Vero cells, which places limitations on their usefulness for enhanced vaccine production. Here, we present a strategy for the development of vaccine seed viruses with enhanced growth in Vero cells by changing an amino acid residue in the stem region of the HA2 subunit of the hemagglutinin (HA) molecule. This mutation optimized the pH for HA-mediated membrane fusion in Vero cells and enhanced virus growth 100 to 1,000 times in the cell line, providing a promising strategy for cell culture-based influenza vaccines.

  20. Determination of ammonia by designing an optode based on immobilization of a Co(III)-Schiff base complex on a triacetylcelluose membrane.

    PubMed

    Tavallali, Hossein; Torabi, Susan; Asadi, Mozaffar; Mohammadi, Hajar

    2014-01-01

    A novel ammonia optode was designed using [4-methyl-N,N'-bis(salicylidene)-1,2-phenylenediamine] tributylphosphincobalt(III) perchlorate monohydrate [Co(4-MeSaloph)(PBu)3]+ complex coated on a triacetylcellulose membrane. The sensor could be used at a wavelength of 378 nm to detect ammonia in the range of 8.3 x 10(-4) and -1.25 x 10(-2) M in borate buffer (pH 10). The LOD was found to be 9.5 x 10(-5) M. The response time of the optode was 2-3 min and depended on the concentration of ammonia. This optode was successfully applied for the determination of ammonia in drinking water.

  1. Characterization of the interaction of hemolytic lectin CEL-III from the marine invertebrate, Cucumaria echinata, with artificial lipid membranes: involvement of neutral sphingoglycolipids in the pore-forming process.

    PubMed

    Hatakeyama, T; Sato, T; Taira, E; Kuwahara, H; Niidome, T; Aoyagi, H

    1999-02-01

    The hemolytic lectin, CEL-III, is a Ca2+-dependent, galactose/N-acetylgalactosamine-specific lectin purified from the marine invertebrate, Cucumaria echinata (Holothuroidea). After binding to specific carbohydrates on the erythrocyte surface, CEL-III forms ion-permeable pores by oligomerizing in the membrane, which leads to colloid osmotic rupture of the cells. When incubated with liposomes composed of total lipids from the human erythrocyte membrane, CEL-III efficiently induced the leakage of carboxyfluorescein (CF) trapped in the vesicles, suggesting the presence of its receptor in the membrane lipids. The rate of CF-leakage increased with increasing temperature, although the hemolytic activity of CEL-III had been found to be much higher at lower temperatures (around 10 degrees C). Identification of the receptor for CEL-III was performed by examining the ability of individual lipids from human erythrocytes to induce CF-leakage from DOPC-liposomes. As a result, the most effective receptor was found to be lactosyl ceramide (LacCer), while globoside (Gb4Cer) also showed slight induction of CF-leakage. On the other hand, a binding assay involving CEL-III-horseradish peroxidase conjugate indicated that CEL-III exhibits similar affinity for LacCer and Gb4Cer, suggesting that the structure or length of the carbohydrate portion of sphingoglycolipids is also relevant as to their ability to induce CF-leakage in addition to their affinity. Electron micrographs of CEL-III-treated liposomes revealed that CEL-III induced considerable morphological changes in the vesicles, while a clearly distinguishable oligomeric structure of the protein was not observed.

  2. Immunogens Modeling a Fusion-Intermediate Conformation of gp41 Elicit Antibodies to the Membrane Proximal External Region of the HIV Envelope Glycoprotein.

    PubMed

    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.

  3. Spectrophotometric method for the determination of ascorbic acid with iron (III)-1,10-phenanthroline after preconcentration on an organic solvent-soluble membrane filter.

    PubMed

    Gu, X; Chen, C; Thou, T

    1996-04-01

    A solvent-soluble membrane filter is proposed for the simple and rapid preconcentration and spectrophotometric determination of ascorbic acid based on the reduction of 1, 10-phenanthroline (phen)-iron (III), which is collected on a nitrocellulose membrane filter as an ion-associate of the cationic complex of tri,phen-iron (II) [ferroin, Fe(phen)(2+)(3)] with an anionic surfactant (of dodecyl sulfate). The ion-associate collected is dissolved in a small volume of 2-methoxyethanol together with the filter. The colour intensity is measured at 510 nm against the reagent blank and is proportional to the content of ascorbic acid in the range 2.5-50 microg ascorbic acid in 5 ml of solvent with excellent reproducibility (RSD 3.2% for 200 microg 1(-1) ascorbic acid), the enrichment factor achieves 100-fold and detection limits better than 2.0 microg 1(-1) can be obtained. Diverse components of organic and inorganic compounds normally present in fruits, vegetable, beverages and urine do not interfere. The recoveries of the ascorbic acid added to the samples are quantitative.

  4. ExsB Is Required for Correct Assembly of the Pseudomonas aeruginosa Type III Secretion Apparatus in the Bacterial Membrane and Full Virulence In Vivo

    PubMed Central

    Perdu, Caroline; Huber, Philippe; Bouillot, Stéphanie; Blocker, Ariel; Elsen, Sylvie; Attrée, Ina

    2015-01-01

    Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa. Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon. PMID:25690097

  5. 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

  6. Microwaves and the cell membrane. III. Protein shedding is oxygen and temperature dependent: evidence for cation bridge involvement

    SciTech Connect

    Liburdy, R.P.; Vanek, P.F. Jr.

    1987-03-01

    Microwaves (2450 MHz, 60 mW/g) are shown to result in the release or shedding of at least 11 low-molecular-weight proteins (less than or equal to 31,000 Da) from rabbit erythrocytes maintained in physiological buffer. Protein release was detected by gel electrophoresis of cell-free supernatants using sensitive silver staining. This release is oxygen dependent and occurs in 30 min for exposures conducted within the special temperature region of 17-21 degrees C, which is linked to a structural or conformational transition in the cell membrane. Shedding of 26,000 and 24,000 Da proteins is unique to microwave treatment, with enhanced release of 28,000 and less than or equal to 15,000 Da species during microwave compared to sham exposures. Two-dimensional isoelectric focusing further reveals that proteins of less than or equal to 14,000 Da shed during microwave treatment exhibit a pI of 6.8-7.3 not seen in sham-treated cells. Treatment of erythrocytes with a serine-directed protease inhibitor does not prevent release of proteins. However, when erythrocytes are maintained at 17-21 degrees C by conventional heating in the absence of divalent cations, release of 28,000-31,000 and less than or equal to 14,000 Da components is detected. This indicates that cation-bridge stability may be important for release of these proteins. The above results provide evidence that microwaves alter erythrocyte protein composition at temperatures linked to a transition in the cell membrane and that destabilization of salt bridges may play a role in an interaction mechanism for protein release.

  7. A Rare Presentation of Os Odontoideum with Multiple Vertebral Fusion in Type III Klippel-Feil Syndrome (KFS) - A Case Report.

    PubMed

    Begum, Shabana; Roy, Tanushree; Ghosal Sen, Tanwi; Gupta, Indrajit

    2014-09-01

    An abnormal atlas and axis with presence of os odontoideum and fusion of multiple vertebrae were noted in an intact skeleton, in the osteology museum of the Department of Anatomy of North Bengal Medical College, West Bengal, India.These multiple abnormalities at various levels along with increased thickness of antero-posterior arch of atlas pointed towards the congenital nature of the anomalies, possibly due to Klippel-Feil syndrome (KFS). These unusual findings denote a developmental background of the manifestations.The cervical instability, resultant neurodeficit and impairment of quality of life of the affected individuals, which are inherent in such cases, reveal their clinical importance.

  8. Involvement of cytoskeletal proteins in the barrier function of the human erythrocyte membrane. III. Permeability of spectrin-depleted inside-out membrane vesicles to hydrophilic nonelectrolytes. Formation of leaks by chemical or enzymatic modification of membrane proteins.

    PubMed

    Klonk, S; Deuticke, B

    1992-04-29

    Spectrin-depleted inside-out vesicles (IOV's) prepared from human erythrocyte membranes were characterized in terms of size, ground permeability to hydrophilic nonelectrolytes and their sensitivity to modification by SH reagents, DIDS and trypsin. IOV's proved to have the same permeability of their lipid domain to erythritol as native erythrocytes, in contrast to resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)), which have a residual leak. On the other hand, IOV's have a slightly elevated permeability for mannitol and sucrose, nonelectrolytes which are almost (mannitol) or fully (sucrose) impermeant in the native membrane. These increased fluxes, which have a high activation energy and can be stimulated by phloretin, are, however, also much smaller than the corresponding leak fluxes observed in resealed ghosts. In view of these differences, formation of IOV's can be concluded to go along with partial annealing of barrier defects persisting in the erythrocyte membrane after preparation of resealed ghosts. Oxidation of SH groups of the IOV membrane by diamide produces an enhancement of permeability for hydrophilic nonelectrolytes which is much less pronounced than that induced by a similar treatment of erythrocytes or ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 126-136 (Part I in this series)). Moreover, proteolytic treatment of the vesicle membrane, although leading to a marked digestion of integral membrane proteins, only induces a minor, saturating increase of permeability, much lower than that in trypsinized resealed ghosts (Klonk, S. and Deuticke, B. (1992) Biochim. Biophys. Acta 1106, 137-142 (Part II of this series)). Since absence of the cytoskeletal proteins, spectrin and actin, is the major difference between IOV's and resealed ghosts, these results may be taken as further evidence for a dependence of the barrier properties of the erythrocyte membrane bilayer domain

  9. A review of data fusion techniques.

    PubMed

    Castanedo, Federico

    2013-01-01

    The integration of data and knowledge from several sources is known as data fusion. This paper summarizes the state of the data fusion field and describes the most relevant studies. We first enumerate and explain different classification schemes for data fusion. Then, the most common algorithms are reviewed. These methods and algorithms are presented using three different categories: (i) data association, (ii) state estimation, and (iii) decision fusion.

  10. A Review of Data Fusion Techniques

    PubMed Central

    2013-01-01

    The integration of data and knowledge from several sources is known as data fusion. This paper summarizes the state of the data fusion field and describes the most relevant studies. We first enumerate and explain different classification schemes for data fusion. Then, the most common algorithms are reviewed. These methods and algorithms are presented using three different categories: (i) data association, (ii) state estimation, and (iii) decision fusion. PMID:24288502

  11. Carbon Nanotubes Mediate Fusion of Lipid Vesicles.

    PubMed

    Bhaskara, Ramachandra M; Linker, Stephanie M; Vögele, Martin; Köfinger, Jürgen; Hummer, Gerhard

    2017-02-28

    The fusion of lipid membranes is opposed by high energetic barriers. In living organisms, complex protein machineries carry out this biologically essential process. Here we show that membrane-spanning carbon nanotubes (CNTs) can trigger spontaneous fusion of small lipid vesicles. In coarse-grained molecular dynamics simulations, we find that a CNT bridging between two vesicles locally perturbs their lipid structure. Their outer leaflets merge as the CNT pulls lipids out of the membranes, creating an hourglass-shaped fusion intermediate with still intact inner leaflets. As the CNT moves away from the symmetry axis connecting the vesicle centers, the inner leaflets merge, forming a pore that completes fusion. The distinct mechanism of CNT-mediated membrane fusion may be transferable, providing guidance in the development of fusion agents, e.g., for the targeted delivery of drugs or nucleic acids.

  12. 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.

  13. A novel fusion protein domain III-capsid from dengue-2, in a highly aggregated form, induces a functional immune response and protection in mice.

    PubMed

    Valdés, Iris; Bernardo, Lidice; Gil, Lázaro; Pavón, Alekis; Lazo, Laura; López, Carlos; Romero, Yaremis; Menendez, Ivón; Falcón, Viviana; Betancourt, Lázaro; Martín, Jorge; Chinea, Glay; Silva, Ricardo; Guzmán, María G; Guillén, Gerardo; Hermida, Lisset

    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(+) and CD8(+) 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.

  14. 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

  15. 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.

  16. Spinal Fusion

    MedlinePlus

    ... concept of fusion is similar to that of welding in industry. Spinal fusion surgery, however, does not ... bone taken from the patient has a long history of use and results in predictable healing. Autograft ...

  17. Ultrastructural Analysis of Myoblast Fusion in Drosophila

    PubMed Central

    Zhang, Shiliang; Chen, Elizabeth H.

    2015-01-01

    Summary Myoblast fusion in Drosophila has become a powerful genetic system with which to unravel the mechanisms underlying cell fusion. The identification of important components of myoblast fusion by genetic analysis has led to a molecular pathway toward our understanding of this cellular process. In addition to the application of immunohistochemistry and live imaging techniques to visualize myoblast fusion at the light microscopic level, ultrastructural analysis using electron microscopy remains an indispensable tool to reveal fusion intermediates and specific membrane events at sites of fusion. In this chapter, we describe conventional chemical fixation and high-pressure freezing/freeze substitution methods for visualizing fusion intermediates during Drosophila myoblast fusion. Furthermore, we describe an immunoelectron microscopic method for localizing specific proteins relative to the fusion apparatus. PMID:18979250

  18. A pH dependent transport and back transport of americium(III) through the cellulose triacetate composite polymer membrane of cyanex-301 and TBP: role of H-bonding interactions.

    PubMed

    Bhattacharyya, Arunasis; Mohapatra, Prasanta Kumar; Ghanty, Tapan Kumar; Manchanda, Vijay Kumar

    2008-11-07

    Transport of Am(III) was studied through the composite polymer membrane of Cyanex-301 [bis(2,4,4-trimethylpentyl)dithiophosphinic acid] and tri-n-butylphosphate (TBP). Depending on the pH of the strip solution containing alpha-hydroxyisobutyric acid (AHIBA), the transport behaviour of Am(III) was changed significantly. After approximately 70% of the Am(III) transported to the strip side, interestingly, back transport of Am(III) was observed at a pH of 3.5. The back transport phenomenon was not so significant at pH 1 and 5.7. The back transport of Am(III) was attributed to the transport of AHIBA from strip to the feed side due to its interaction with TBP in the membrane and the attainment of Donnan equilibrium because of the presence of Na(+) in the feed as the driving ion. The experimental observations were rationalized using the hydrogen bonding interaction energies obtained through ab initio molecular orbital and DFT calculations.

  19. Generalized Chernoff Fusion Approximation for Practical Distributed Data Fusion

    DTIC Science & Technology

    2009-07-01

    Generalized Chernoff Fusion Approximation for Practical Distributed Data Fusion William J. Farrell III R&D Department Adaptive Methods , Inc...independence or modify legacy systems with pedigree tagging techniques . Leveraging the well- known Covariance Intersection algorithm, its generalization...Adaptive Methods , Inc.,Centreville, VA , , 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR

  20. Radioscapholunate Fusions

    PubMed Central

    McGuire, Duncan Thomas; Bain, Gregory Ian

    2012-01-01

    Radiocarpal fusions are performed for a variety of indications, most commonly for debilitating painful arthritis. The goal of a wrist fusion is to fuse the painful, diseased joints and to preserve motion through the healthy joints. Depending on the extent of the disease process, radiocarpal fusions may take the form of radiolunate, radioscapholunate, or total wrist fusions. Surgical techniques and instrumentation have advanced over the last few decades, and consequently the functional outcomes have improved and complications decreased. Techniques for partial carpal fusions have improved and now include distal scaphoid and triquetrum excision, which improves range of motion and fusion rates. In this article we discuss the various surgical techniques and fixation methods available and review the corresponding evidence in the literature. The authors' preferred surgical technique of radioscapholunate fusion with distal scaphoid and triquetrum excision is outlined. New implants and new concepts are also discussed. PMID:24179717

  1. Controlled cellular fusion using optically trapped plasmonic nano-heaters

    NASA Astrophysics Data System (ADS)

    Bahadori, Azra; Lund, Andreas R.; Semsey, Szabolcs; Oddershede, Lene B.; Bendix, Poul M.

    2016-09-01

    Optically trapped plasmonic nano-heaters are used to mediate efficient and controlled fusion of biological membranes. The fusion method is demonstrated by optically trapping plasmonic nanoparticles located in between vesicle membranes leading to rapid lipid and content mixing. As an interesting application we show how direct control over fusion can be used for studying diffusion of peripheral membrane proteins and their interactions with membranes and for studying protein reactions. Membrane proteins encapsulated in an inert vesicle can be transferred to a vesicle composed of negative lipids by optically induced fusion. Mixing of the two membranes results in a fused vesicle with a high affinity for the protein and we observe immediate membrane tubulation due to the activity of the protein. Fusion of distinct membrane compartments also has applications in small scale chemistry for realizing pico-liter reactions and offers many exciting applications within biology which are discussed here.

  2. 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.

  3. HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain.

    PubMed

    Lu, Lu; Tong, Pei; Yu, Xiaowen; Pan, Chungen; Zou, Peng; Chen, Ying-Hua; Jiang, Shibo

    2012-12-01

    Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors.

  4. Class III PI 3-kinase is the main source of PtdIns3P substrate and membrane recruitment signal for PIKfyve constitutive function in podocyte endomembrane homeostasis.

    PubMed

    Ikonomov, Ognian C; Sbrissa, Diego; Venkatareddy, Madhusudan; Tisdale, Ellen; Garg, Puneet; Shisheva, Assia

    2015-05-01

    The evolutionarily conserved PIKfyve, which synthesizes PtdIns5P from PtdIns, and PtdIns(3,5)P2 from PtdIns3P, requires PtdIns3P as both an enzyme substrate and a membrane recruitment signal. Whereas the PtdIns3P source is undetermined, class III PI3K (Vps34), the only evolutionarily conserved of the eight mammalian PI3Ks, is presumed as a main candidate. A hallmark of PIKfyve deficiency is formation of multiple translucent cytoplasmic vacuoles seen by light microscopy in cells cultured in complete media. Such an aberrant phenotype is often observed in cells from conditional Vps34 knockout (KO) mice. To clarify the mechanism of Vps34 KO-triggered vacuolation and the PtdIns3P source for PIKfyve functionality, here we have characterized a podocyte cell type derived from Vps34fl/fl mice, which, upon Cre-mediated gene KO, robustly formed cytoplasmic vacuoles resembling those in PikfyveKO MEFs. Vps34wt, expressed in Vps34KO podocytes restored the normal morphology, but only if the endogenous PIKfyve activity was intact. Conversely, expressed PIKfyvewt rescued completely the vacuolation only in PikfyveKO MEFs but not in Vps34KO podocytes. Analyses of phosphoinositide profiles by HPLC and localization patterns by a PtdIns3P biosensor revealed that Vps34 is the main supplier of localized PtdIns3P not only for PIKfyve activity but also for membrane recruitment. Concordantly, Vps34KO podocytes had severely reduced steady-state levels of both PtdIns(3,5)P2 and PtdIns5P, along with PtdIns3P. We further revealed a plausible physiologically-relevant Vps34-independent PtdIns3P supply for PIKfyve, operating through activated class I PI3Ks. Our data provide the first evidence that the vacuolation phenotype in Vps34KO podocytes is due to PIKfyve dysfunction and that Vps34 is a main PtdIns3P source for constitutive PIKfyve functionality.

  5. Fusion breeder

    SciTech Connect

    Moir, R.W.

    1982-04-20

    The fusion breeder is a fusion reactor designed with special blankets to maximize the transmutation by 14 MeV neutrons of uranium-238 to plutonium or thorium to uranium-233 for use as a fuel for fission reactors. Breeding fissile fuels has not been a goal of the US fusion energy program. This paper suggests it is time for a policy change to make the fusion breeder a goal of the US fusion program and the US nuclear energy program. The purpose of this paper is to suggest this policy change be made and tell why it should be made, and to outline specific research and development goals so that the fusion breeder will be developed in time to meet fissile fuel needs.

  6. Fusion breeder

    SciTech Connect

    Moir, R.W.

    1982-02-22

    The fusion breeder is a fusion reactor designed with special blankets to maximize the transmutation by 14 MeV neutrons of uranium-238 to plutonium or thorium to uranium-233 for use as a fuel for fission reactors. Breeding fissile fuels has not been a goal of the US fusion energy program. This paper suggests it is time for a policy change to make the fusion breeder a goal of the US fusion program and the US nuclear energy program. The purpose of this paper is to suggest this policy change be made and tell why it should be made, and to outline specific research and development goals so that the fusion breeder will be developed in time to meet fissile fuel needs.

  7. The egg membrane microdomain-associated uroplakin III-Src system becomes functional during oocyte maturation and is required for bidirectional gamete signaling at fertilization in Xenopus laevis.

    PubMed

    Mahbub Hasan, A K M; Hashimoto, Aki; Maekawa, Yuka; Matsumoto, Takashi; Kushima, Shota; Ijiri, Takashi W; Fukami, Yasuo; Sato, Ken-ichi

    2014-04-01

    In Xenopus laevis, sperm-egg interaction promotes partial proteolysis and/or tyrosine phosphorylation of uroplakin III (UPIII) and the tyrosine kinase Src, which both localize to the cholesterol-enriched egg membrane microdomains (MDs). Here we show that sperm promote proteolysis and/or tyrosine phosphorylation of UPIII and Src in MDs isolated from ovulated and unfertilized eggs (UF-MDs). An antibody against the extracellular domain of UPIII interferes with these events. Inhibition of fertilization by anti-UPIII antibody is rescued by co-incubation with UF-MDs. This suggests that, like MDs in intact eggs, the isolated UF-MDs are capable of interacting with sperm, an interaction that does not interfere with normal fertilization but rather augments the ability of sperm to fertilize eggs pretreated with anti-UPIII antibody. This unexpected effect of UF-MDs on sperm requires UPIII function in UF-MDs and protein kinase activity in sperm. MDs isolated from progesterone-treated mature oocytes, but not ovarian immature oocytes, are similarly functional as UF-MDs. The anti-UPIII extracellular domain antibody binds more effectively to the surface of mature than immature ovarian oocytes. We propose that the structural and functional competency of the UPIII-Src signaling system in MDs is strictly regulated during oocyte maturation and subsequently in sperm-mediated egg activation and fertilization. The fertilization-related signaling properties seen in UF-MDs can be partially reconstituted in MDs of human embryonic kidney 293 cells (293-MDs) expressing UPIII, Src and uroplakin Ib. However, 293-MDs expressing a proteolysis-resistant mutant of UPIII are less functional, suggesting that the availability of UPIII to protease action is important for MD function.

  8. Functional homology of gHs and gLs from EBV-related gamma-herpesviruses for EBV-induced membrane fusion.

    PubMed

    Omerović, Jasmina; Longnecker, Richard

    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.

  9. 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.

  10. Inhibition of HIV-1 by fusion inhibitors.

    PubMed

    Eggink, Dirk; Berkhout, Ben; Sanders, Rogier W

    2010-01-01

    The envelope glycoprotein complex (Env) is responsible for entry of the human immunodeficiency virus type 1 (HIV-1) into cells by mediating attachment to target cells and subsequent membrane fusion. Env consists of three gp120 subunits that mediate receptor and co-receptor attachment and three gp41 subunits responsible for membrane fusion. Several steps of the entry process can serve as drug targets. Receptor antagonists prevent attachment of gp120 to the receptor or co-receptor and conformational changes within gp41 required for membrane fusion can be inhibited by fusion inhibitors. Enfuvirtide (T20, Fuzeon) is a peptide based on the gp41 sequence and is the only approved fusion inhibitor. It prevents membrane fusion by competitively binding to gp41 and blocking the formation of the post-fusion structure. New generations of T20-like peptides have been developed with improved potency and stability. Besides T20 and derivatives, other fusion inhibitors have been developed that target different domains of gp41. Here we discuss the development of fusion inhibitors, their mode of action and their potential for incorporation in future drug regimens.

  11. Mechanisms of myoblast fusion during muscle development

    PubMed Central

    Kim, Ji Hoon; Jin, Peng; Duan, Rui; Chen, Elizabeth H.

    2015-01-01

    The development and regeneration of skeletal muscles require the fusion of mononulceated muscle cells to form multinucleated, contractile muscle fibers. Studies using a simple genetic model, Drosophila melanogaster, have discovered many evolutionarily conserved fusion-promoting factors in vivo. Recent work in zebrafish and mouse also identified several vertebrate-specific factors required for myoblast fusion. Here, we integrate progress in multiple in vivo systems and highlight conceptual advance in understanding how muscle cell membranes are brought together for fusion. We focus on the molecular machinery at the fusogenic synapse and present a three-step model to describe the molecular and cellular events leading to fusion pore formation. PMID:25989064

  12. A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase1[W

    PubMed Central

    Mikami, Koji; Saavedra, Laura; Hiwatashi, Yuji; Uji, Toshiki; Hasebe, Mitsuyasu; Sommarin, Marianne

    2010-01-01

    Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific. PMID:20427464

  13. Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?).

    PubMed Central

    De Michelis, M. I.; Rasi-Caldogno, F.; Pugliarello, M. C.; Olivari, C.

    1996-01-01

    A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase. PMID:12226232

  14. 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.

  15. Rare earth ions block the ion pores generated by the class II fusion proteins of alphaviruses and allow analysis of the biological functions of these pores.

    PubMed

    Koschinski, Andreas; Wengler, Gerd; Wengler, Gisela; Repp, Holger

    2005-12-01

    Recently, class II fusion proteins have been identified on the surface of alpha- and flaviviruses. These proteins have two functions besides membrane fusion: they generate an isometric lattice on the viral surface and they form ion-permeable pores at low pH. An attempt was made to identify inhibitors for the ion pores generated by the fusion proteins of the alphaviruses Semliki Forest virus and Sindbis virus. These pores can be detected and analysed in three situations: (i) in the target membrane during virus entry, by performing patch-clamp measurements of membrane currents; (ii) in the virus particle, by studying the entry of propidium iodide; and (iii) in the plasma membrane of infected cells, by Fura-2 fluorescence imaging of Ca2+ entry into infected cells. It is shown here that, at a concentration of 0.1 mM, rare earth ions block the ion permeability of alphavirus ion pores in all three situations. Even at a concentration of 0.5 mM, these ions do not block formation of the viral fusion pore, as they do not inhibit entry or multiplication of alphaviruses. The data indicate that ions flow through the ion pores into the virus particle in the endosome and from the endosome into the cytoplasm after fusion of the viral envelope with the endosomal membrane. These ion flows, however, are not necessary for productive infection. The possibility that the ability of class II fusion proteins to form ion-permeable pores reflects their origin from protein toxins that form ion-permeable pores, and that entry via class II fusion proteins may resemble the entry of non-enveloped viruses, is discussed.

  16. 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

  17. 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

  18. 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.

  19. Control of mechanically activated polymersome fusion: Factors affecting fusion

    DOE PAGES

    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

  20. 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.

  1. 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)

  2. 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.

  3. Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

    PubMed

    Yan, X; Gonzales, R A; Wagner, G J

    1997-11-01

    Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.

  4. Gene fusions of signal sequences with a modified beta-glucuronidase gene results in retention of the beta-glucuronidase protein in the secretory pathway/plasma membrane.

    PubMed Central

    Yan, X; Gonzales, R A; Wagner, G J

    1997-01-01

    Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM. PMID:9390428

  5. Modes of Paramyxovirus Fusion: a Henipavirus perspective

    PubMed Central

    Lee, Benhur; Akyol-Ataman, Zeynep

    2011-01-01

    Henipavirus is a new genus of paramyxovirus that uses protein-based receptors (EphrinB2 and EphrinB3) for virus entry. Paramyxovirus entry requires the coordinated action of the fusion (F) and attachment viral envelope glycoproteins. Receptor binding to the attachment protein triggers F to undergo a conformational cascade that results in membrane fusion. The accumulation of structural and functional studies on many paramyxoviral fusion and attachment proteins, including recent structures of Nipah and Hendra virus G bound and unbound to cognate ephrinB receptors, indicate that henipavirus entry and fusion differs mechanistically from paramyxoviruses that use glycan-based receptors. PMID:21511478

  6. 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)

  7. 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.

  8. Hendra virus fusion protein transmembrane domain contributes to pre-fusion protein stability.

    PubMed

    Webb, Stacy; Nagy, Tamas; Moseley, Hunter; Fried, Michael; Dutch, Rebecca Ellis

    2017-02-17

    Enveloped viruses utilize fusion (F) proteins studding the surface of the virus to facilitate membrane fusion with a target cell membrane. Fusion of the viral envelope with a cellular membrane is required for release of viral genomic material so the virus can ultimately reproduce and spread. To drive fusion, the F protein undergoes an irreversible conformational change, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Mutations in F protein transmembrane (TM) domains implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was utilized to demonstrate that isolated TM domains of Hendra virus F protein associate in a monomer-trimer equilibrium (Smith EC, et al. Trimeric transmembrane domain interactions in paramyxovirus fusion proteins. 2013. J Biol Chem. 288, 35726). To determine factors driving this association, 140 paramyxovirus F protein TM domain sequences were analyzed. A heptad repeat of β-branched residues was found and analysis of the Hendra virus F TM domain revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein stability, including pre-fusion conformation stability. Together our data suggest that the heptad repeat LIZ contributed to TM-TM association and is important for F protein function and pre-fusion stability.

  9. Deduced sequences of the membrane fusion and attachment proteins of canine distemper viruses isolated from dogs and wild animals in Korea.

    PubMed

    Bae, Chae-Wun; Lee, Joong-Bok; Park, Seung-Yong; Song, Chang-Seon; Lee, Nak-Hyung; Seo, Kun-Ho; Kang, Young-Sun; Park, Choi-Kyu; Choi, In-Soo

    2013-08-01

    Canine distemper virus (CDV) causes highly contagious respiratory, gastrointestinal, and neurological diseases in wild and domestic animal species. Despite a broad vaccination campaign, the disease is still a serious problem worldwide. In this study, six field CDV strains were isolated from three dogs, two raccoon dogs, and one badger in Korea. The full sequence of the genes encoding fusion (F) and hemagglutinin (H) proteins were compared with those of other CDVs including field and vaccine strains. The phylogenetic analysis for the F and H genes indicated that the two CDV strains isolated from dogs were most closely related to Chinese strains in the Asia-1 genotype. Another four strains were closely related to Japanese strains in the Asia-2 genotype. The six currently isolated strains shared 90.2-92.1% and 88.2-91.8% identities with eight commercial vaccine strains in their nucleotide and amino acid sequences of the F protein, respectively. They also showed 90.1-91.4% and 87.8-90.7% identities with the same vaccine strains in their nucleotide and deduced amino acid sequences of the H protein, respectively. Different N-linked glycosylation sites were identified in the F and H genes of the six isolates from the prototype vaccine strain Onderstepoort. Collectively, these results demonstrate that at least two different CDV genotypes currently exist in Korea. The considerable genetic differences between the vaccine strains and wild-type isolates would be a major factor of the incomplete protection of dogs from CDV infections.

  10. A review of reagents for fluorescence microscopy of cellular compartments and structures, Part III: reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm.

    PubMed

    Kilgore, Jason A; Dolman, Nick J; Davidson, Michael W

    2014-01-02

    Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image.

  11. 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

    2013-01-02

    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.

  12. Signaling mechanisms in mammalian myoblast fusion.

    PubMed

    Hindi, Sajedah M; Tajrishi, Marjan M; Kumar, Ashok

    2013-04-23

    Myoblast fusion is a critical process that contributes to the growth of muscle during development and to the regeneration of myofibers upon injury. Myoblasts fuse with each other as well as with multinucleated myotubes to enlarge the myofiber. Initial studies demonstrated that myoblast fusion requires extracellular calcium and changes in cell membrane topography and cytoskeletal organization. More recent studies have identified several cell-surface and intracellular proteins that mediate myoblast fusion. Furthermore, emerging evidence suggests that myoblast fusion is also regulated by the activation of specific cell-signaling pathways that lead to the expression of genes whose products are essential for the fusion process and for modulating the activity of molecules that are involved in cytoskeletal rearrangement. Here, we review the roles of the major signaling pathways in mammalian myoblast fusion.

  13. Spinal fusion

    MedlinePlus

    Liu G, Wong HK. Laminectomy and fusion. In: Shen FH, Samartzis D, Fessler RG, eds. Textbook of the Cervical Spine . Philadelphia, PA: Elsevier Saunders; 2015:chap 34. Wood GW. Arthrodesis of the spine. In: Canale ST, Beaty JH, eds. Campbell's Operative ...

  14. 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

  15. Robust Expression of the Human Neonatal Fc Receptor in a Truncated Soluble Form and as a Full-Length Membrane-Bound Protein in Fusion with eGFP

    PubMed Central

    Seijsing, Johan; Lindborg, Malin; Löfblom, John; Uhlén, Mathias; Gräslund, Torbjörn

    2013-01-01

    Studies on the neonatal Fc receptor (FcRn) have revealed a multitude of important functions in mammals, including protection of IgG and serum albumin (SA) from lysosomal degradation. The pharmacokinetic behavior of therapeutic antibodies, IgG-Fc- and SA-containing drugs is therefore influenced by their interaction with FcRn. Pre-clinical development of such drugs is facilitated if their interaction with FcRn can be studied in vitro. For this reason we have developed a robust system for production of the soluble extracellular domain of human FcRn as well as the full-length receptor as fusion to green fluorescent protein, taking advantage of a lentivirus-based gene delivery system where stable over-expressing cells are easily and rapidly generated. Production of the extracellular domain in multiple-layered culture flasks, followed by affinity purification using immobilized IgG, resulted in capture of milligram amounts of soluble receptor per liter cell culture with retained IgG binding. The receptor was further characterized by SDS-PAGE, western blotting, circular dichroism spectroscopy, ELISA, surface plasmon resonance and a temperature stability assay showing a functional and stable protein of high purity. The full-length receptor was found to be successfully over-expressed in a membrane-bound form with retained pH-dependent IgG- and SA-binding. PMID:24260574

  16. 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.

  17. Moesin is required for HIV-1-induced CD4-CXCR4 interaction, F-actin redistribution, membrane fusion and viral infection in lymphocytes.

    PubMed

    Barrero-Villar, Marta; Cabrero, José Román; Gordón-Alonso, Mónica; Barroso-González, Jonathan; Alvarez-Losada, Susana; Muñoz-Fernández, M Angeles; Sánchez-Madrid, Francisco; Valenzuela-Fernández, Agustín

    2009-01-01

    The human immunodeficiency virus 1 (HIV-1) envelope regulates the initial attachment of viral particles to target cells through its association with CD4 and either CXCR4 or CCR5. Although F-actin is required for CD4 and CXCR4 redistribution, little is known about the molecular mechanisms underlying this fundamental process in HIV infection. Using CD4(+) CXCR4(+) permissive human leukemic CEM T cells and primary lymphocytes, we have investigated whether HIV-1 Env might promote viral entry and infection by activating ERM (ezrin-radixin-moesin) proteins to regulate F-actin reorganization and CD4/CXCR4 co-clustering. The interaction of the X4-tropic protein HIV-1 gp120 with CD4 augments ezrin and moesin phosphorylation in human permissive T cells, thereby regulating ezrin-moesin activation. Moreover, the association and clustering of CD4-CXCR4 induced by HIV-1 gp120 requires moesin-mediated anchoring of actin in the plasma membrane. Suppression of moesin expression with dominant-negative N-moesin or specific moesin silencing impedes reorganization of F-actin and HIV-1 entry and infection mediated by the HIV-1 envelope protein complex. Therefore, we propose that activated moesin promotes F-actin redistribution and CD4-CXCR4 clustering and is also required for efficient X4-tropic HIV-1 infection in permissive lymphocytes.

  18. Role of the synaptobrevin C terminus in fusion pore formation

    PubMed Central

    Ngatchou, Annita N.; Kisler, Kassandra; Fang, Qinghua; Walter, Alexander M.; Zhao, Ying; Bruns, Dieter; Sørensen, Jakob B.; Lindau, Manfred

    2010-01-01

    Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets. PMID:20937897

  19. 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

  20. 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