WorldWideScience

Sample records for bilayer membrane fusion

  1. Dolichol phosphate induces non-bilayer structures, vesicle fusion and transbilayer movements of lipids in model membranes

    Energy Technology Data Exchange (ETDEWEB)

    de Kruijff, B.; Van Duijn, G.; Valtersson, C.; Chojnacki, T.; Verkleij, A.J.; Dallner, G.

    1987-05-01

    The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichol phosphate on the structure and fluidity of model membranes was studied using different biophysical techniques. These studies suggest that (1) dolichol and dolichol derivatives destabilize unsaturated PE-containing bilayers and promote hexagonal II phase formation; (2) high concentrations of dolichol induce lipid structures characterized by isotropic T P NMR and particulate fracture faces. The effect of dolichol and dolichyl phosphate on fusion between large unilamellar vesicles of DOPC and DOPE was studied using a fluroescence resonance energy transfer assay. The influence of dolichyl phosphate on the transbilary movement of DOPC in multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) composed of DOPC and DOPE (1:2) was investigated by using the PC-specified transfer protein. The results indicate that: (1) both dolichol and dolichyl phosphate enhance vesicle fusion in a comparable and concentration-dependent way; (2) the amount of exchangeable PC from MLVs is increased by dolichyl phosphate probably as a result of fusion processes. It thus appears that these polyprenols are potent destabilizers of bilayer structure and that this process is accompanied by membrane fusion and transbilayer transport of phospholipids.

  2. Dolichol phosphate induces non-bilayer structures, vesicle fusion and transbilayer movements of lipids in model membranes

    International Nuclear Information System (INIS)

    The effect of dolichols, polyprenols, dolichol esterified with fatty acids, and dolichol phosphate on the structure and fluidity of model membranes was studied using different biophysical techniques. These studies suggest that (1) dolichol and dolichol derivatives destabilize unsaturated PE-containing bilayers and promote hexagonal II phase formation; (2) high concentrations of dolichol induce lipid structures characterized by isotropic 31P NMR and particulate fracture faces. The effect of dolichol and dolichyl phosphate on fusion between large unilamellar vesicles of DOPC and DOPE was studied using a fluroescence resonance energy transfer assay. The influence of dolichyl phosphate on the transbilary movement of DOPC in multilamellar vesicles (MLV) and large unilamellar vesicles (LUV) composed of DOPC and DOPE (1:2) was investigated by using the PC-specified transfer protein. The results indicate that: (1) both dolichol and dolichyl phosphate enhance vesicle fusion in a comparable and concentration-dependent way; (2) the amount of exchangeable PC from MLVs is increased by dolichyl phosphate probably as a result of fusion processes. It thus appears that these polyprenols are potent destabilizers of bilayer structure and that this process is accompanied by membrane fusion and transbilayer transport of phospholipids

  3. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  4. Viral membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-05-15

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

  5. Viral membrane fusion

    International Nuclear Information System (INIS)

    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. The role of cholesterol in membrane fusion.

    Science.gov (United States)

    Yang, Sung-Tae; Kreutzberger, Alex J B; Lee, Jinwoo; Kiessling, Volker; Tamm, Lukas K

    2016-09-01

    Cholesterol modulates the bilayer structure of biological membranes in multiple ways. It changes the fluidity, thickness, compressibility, water penetration and intrinsic curvature of lipid bilayers. In multi-component lipid mixtures, cholesterol induces phase separations, partitions selectively between different coexisting lipid phases, and causes integral membrane proteins to respond by changing conformation or redistribution in the membrane. But, which of these often overlapping properties are important for membrane fusion?-Here we review a range of recent experiments that elucidate the multiple roles that cholesterol plays in SNARE-mediated and viral envelope glycoprotein-mediated membrane fusion. PMID:27179407

  7. Fusion and fission of fluid amphiphilic bilayers.

    Science.gov (United States)

    Gotter, Martin; Strey, Reinhard; Olsson, Ulf; Wennerström, Håkan

    2005-01-01

    The system water-oil (n-decane)-nonionic surfactant (C12E5) forms bilayer phases in a large concentration region, but, for a given oil-to-surfactant ratio, only in a narrow temperature range. In addition to the anisotropic lamellar phase (Lalpha) there is also, at slightly higher temperature, a sponge or L3-phase where the bilayers build up an isotropic structure extending macroscopically in three dimensions. In this phase the bilayer mid-surface has a mean curvature close to zero and a negative Euler characteristic. In this paper we study how the bilayers in the lamellar and the sponge phase respond dynamically to sudden temperature changes. The monolayer spontaneous curvature depends sensitively on temperature and a change of temperature thus provides a driving force for a change in bilayer topology. The equilibration therefore involves kinetic steps of fusion/fission of bilayers. Such dynamic processes have previously been monitored by temperature jump experiments using light scattering in the sponge phase. These experiments revealed an extraordinarily strong dependence of the relaxation time on the bilayer volume fraction phi. At phi magnetic resonance (2H-NMR) appear feasible. We here report on the first experiments concerned with the dynamics of the macroscopic phase transition sponge-lamellae by 2H-NMR. We find that the sponge-to-lamellae transition occurs through a nucleation process followed by domain growth involving bilayer fission at domain boundaries. In contrast, the lamellae-to-sponge transformation apparently occurs through a succession of uncorrelated bilayer fusion events. PMID:15715316

  8. The lipidic particle as an intermediate structure in membrane fusion processes and bilayer to hexagonal HII transitions

    OpenAIRE

    Verkleij, A.J.; Echteld, C.J.A. van; Gerritsen, W.J.; Cullis, P.R.; de Kruijff, B.

    1980-01-01

    Small unilamellar vesicles comprised of a mixture of phosphatidylethanolamine/phosphatidylcholine/cholesterol (3 : 1 : 2) fuse to form large multilamellar vesicles on increasing the temperature from 0 to 50°C. This event is associated with the appearance of lipidic particles at the fusion sites, consistent with a role as intermediary structures during the fusion process. Further, for phosphatidylcholine/cardiolipin (1 : 1) liposomes in the presence of Mn2+ a direct relationship between lipidi...

  9. Mechanical tension drives cell membrane fusion

    OpenAIRE

    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.

    2015-01-01

    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 inv...

  10. Fusion of biological membranes

    Indian Academy of Sciences (India)

    K Katsov; M Müller; M Schick

    2005-06-01

    The process of membrane fusion has been examined by Monte Carlo simulation, and is found to be very different than the conventional picture. The differences in mechanism lead to several predictions, in particular that fusion is accompanied by transient leakage. This prediction has recently been verified. Self-consistent field theory is applied to examine the free energy barriers in the different scenarios.

  11. DNA nanostructures interacting with lipid bilayer membranes.

    Science.gov (United States)

    Langecker, Martin; Arnaut, Vera; List, Jonathan; Simmel, Friedrich C

    2014-06-17

    CONSPECTUS: DNA has been previously shown to be useful as a material for the fabrication of static nanoscale objects, and also for the realization of dynamic molecular devices and machines. In many cases, nucleic acid assemblies directly mimic biological structures, for example, cytoskeletal filaments, enzyme scaffolds, or molecular motors, and many of the applications envisioned for such structures involve the study or imitation of biological processes, and even the interaction with living cells and organisms. An essential feature of biological systems is their elaborate structural organization and compartmentalization, and this most often involves membranous structures that are formed by dynamic assemblies of lipid molecules. Imitation of or interaction with biological systems using the tools of DNA nanotechnology thus ultimately and necessarily also involves interactions with lipid membrane structures, and thus the creation of DNA-lipid hybrid assemblies. Due to their differing chemical nature, however, highly charged nucleic acids and amphiphilic lipids do not seem the best match for the construction of such systems, and in fact they are rarely found in nature. In recent years, however, a large variety of lipid-interacting DNA conjugates were developed, which are now increasingly being applied also for the realization of DNA nanostructures interacting with lipid bilayer membranes. In this Account, we will present the current state of this emerging class of nanosystems. After a brief overview of the basic biophysical and biochemical properties of lipids and lipid bilayer membranes, we will discuss how DNA molecules can interact with lipid membranes through electrostatic interactions or via covalent modification with hydrophobic moieties. We will then show how such DNA-lipid interactions have been utilized for the realization of DNA nanostructures attached to or embedded within lipid bilayer membranes. Under certain conditions, DNA nanostructures remain mobile on

  12. The Fusion of Membranes and Vesicles: Pathway and Energy Barriers from Dissipative Particle Dynamics

    OpenAIRE

    Grafmüller, Andrea; Shillcock, Julian; Lipowsky, Reinhard

    2009-01-01

    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 configu...

  13. Bilayer-thickness-mediated interactions between integral membrane proteins

    Science.gov (United States)

    Kahraman, Osman; Koch, Peter D.; Klug, William S.; Haselwandter, Christoph A.

    2016-04-01

    Hydrophobic thickness mismatch between integral membrane proteins and the surrounding lipid bilayer can produce lipid bilayer thickness deformations. Experiment and theory have shown that protein-induced lipid bilayer thickness deformations can yield energetically favorable bilayer-mediated interactions between integral membrane proteins, and large-scale organization of integral membrane proteins into protein clusters in cell membranes. Within the continuum elasticity theory of membranes, the energy cost of protein-induced bilayer thickness deformations can be captured by considering compression and expansion of the bilayer hydrophobic core, membrane tension, and bilayer bending, resulting in biharmonic equilibrium equations describing the shape of lipid bilayers for a given set of bilayer-protein boundary conditions. Here we develop a combined analytic and numerical methodology for the solution of the equilibrium elastic equations associated with protein-induced lipid bilayer deformations. Our methodology allows accurate prediction of thickness-mediated protein interactions for arbitrary protein symmetries at arbitrary protein separations and relative orientations. We provide exact analytic solutions for cylindrical integral membrane proteins with constant and varying hydrophobic thickness, and develop perturbative analytic solutions for noncylindrical protein shapes. We complement these analytic solutions, and assess their accuracy, by developing both finite element and finite difference numerical solution schemes. We provide error estimates of our numerical solution schemes and systematically assess their convergence properties. Taken together, the work presented here puts into place an analytic and numerical framework which allows calculation of bilayer-mediated elastic interactions between integral membrane proteins for the complicated protein shapes suggested by structural biology and at the small protein separations most relevant for the crowded membrane

  14. Poxvirus entry and membrane fusion

    International Nuclear Information System (INIS)

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

  15. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion

    DEFF Research Database (Denmark)

    Peters, C; Bayer, M J; Bühler, S;

    2001-01-01

    SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2+/calmodu......SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion of vesicles. But how bilayer mixing--the subsequent core process of fusion--is catalysed remains unclear. Ca2......+/calmodulin controls this terminal process in many intracellular fusion events. Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing membranes form complexes. V0 trans...

  16. Formation of supported lipid bilayers by vesicle fusion

    DEFF Research Database (Denmark)

    Lind, Tania Kjellerup; Cardenas Gomez, Marite; Wacklin, Hanna

    2014-01-01

    We have investigated the effect of deposition temperature on supported lipid bilayer formation via vesicle fusion. By using several complementary surface-sensitive techniques, we demonstrate that despite contradicting literature on the subject, high-quality bilayers can be formed below the main p...... observed during the deposition process by QCM-D correspond to vesicles absorbed on top of a continuous bilayer and not to a surface-supported vesicular layer as previously reported. © 2014 American Chemical Society.......We have investigated the effect of deposition temperature on supported lipid bilayer formation via vesicle fusion. By using several complementary surface-sensitive techniques, we demonstrate that despite contradicting literature on the subject, high-quality bilayers can be formed below the main...... phase-transition temperature of the lipid. We have carefully studied the formation mechanism of supported DPPC bilayers below and above the lipid melting temperature (Tm) by quartz crystal microbalance and atomic force microscopy under continuous flow conditions. We also measured the structure of lipid...

  17. Dynamics and instabilities of lipid bilayer membrane shapes

    OpenAIRE

    Shi, Zheng; Baumgart, Tobias

    2014-01-01

    Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. The lipid bilayer represents the fundamental architecture of the cellular membrane with its shapes determined by the Helfrich curvature bending energy. However, the dynamics of bilayer shape transitions, especially their modulation by membrane proteins, and the resulting shape instabilities, are still not well understood. Here, we review in a unifying manner several theories that descri...

  18. Stalk structures in lipid bilayer fusion studied by x-ray diffraction

    OpenAIRE

    Aeffner, Sebastian

    2012-01-01

    The fusion of two biological membranes is an important step in many processes on the cellular and sub-cellular level. Understanding the involved interplay of different lipid species, a specialized protein machinery and water on length scales of few nanometers poses a significant challenge to current structural biology. Among several complementary approaches, one strategy is to study the structural rearrangements of the lipid matrix. As the initial step, lipid bilayers must be forced into clos...

  19. Detergent interaction with tethered bilayer lipid membranes for protein reconstitution

    Science.gov (United States)

    Broccio, Matteo; Zan Goh, Haw; Loesche, Mathias

    2009-03-01

    Tethered bilayer lipid membranes (tBLMs) are self-assembled biomimetic structures in which the membrane is separated from a solid substrate by a nm-thick hydrated submembrane space. These model systems are being used in binding studies of peripheral proteins and exotoxins. Here we aim at their application for the reconstitution of water-insoluble integral membrane proteins. As an alternative to fusion of preformed proteoliposomes we study the direct reconstitution of such proteins for applications in biosensing and pharmaceutical screening. For reconstitution, highly insulating tBLMs (R˜10^5-10^6 φ) were temporarily incubated with a detergent to screen for conditions that keep the detergent-saturated membranestable and ready to incorporate detergent-solubilized proteins. We assess the electrical characteristics, i.e. specific resistance and capacitance, by means of electrochemical impedance spectroscopy (EIS) under timed incubation with decylmaltoside and dodecylmaltoside detergents in a regime around their critical micelle concentration, 1.8 mM and 0.17 mM respectively and demonstrate the restoration of the tBLM upon detergent removal. Thereby a range of concentration and incubation times was identified, that represents optimal conditions for the subsequent membrane protein reconstitution.

  20. Helix insertion into bilayers and the evolution of membrane proteins

    OpenAIRE

    Renthal, Robert

    2009-01-01

    Polytopic α-helical membrane proteins cannot spontaneously insert into lipid bilayers without assistance from polytopic α-helical membrane proteins that already reside in the membrane. This raises the question of how these proteins evolved. Our current knowledge of the insertion of α-helices into natural and model membranes is reviewed with the goal of gaining insight into the evolution of membrane proteins. Topics include: translocon-dependent membrane protein insertion, antibiotic peptides ...

  1. Dynamics and instabilities of lipid bilayer membrane shapes.

    Science.gov (United States)

    Shi, Zheng; Baumgart, Tobias

    2014-06-01

    Biological membranes undergo constant shape remodeling involving the formation of highly curved structures. The lipid bilayer represents the fundamental architecture of the cellular membrane with its shapes determined by the Helfrich curvature bending energy. However, the dynamics of bilayer shape transitions, especially their modulation by membrane proteins, and the resulting shape instabilities, are still not well understood. Here, we review in a unifying manner several theories that describe the fluctuations (i.e. undulations) of bilayer shapes as well as their local coupling with lipid or protein density variation. The coupling between local membrane curvature and lipid density gives rise to a 'slipping mode' in addition to the conventional 'bending mode' for damping the membrane fluctuation. This leads to a number of interesting experimental phenomena regarding bilayer shape dynamics. More importantly, curvature-inducing proteins can couple with membrane shape and eventually render the membrane unstable. A criterion for membrane shape instability is derived from a linear stability analysis. The instability criterion reemphasizes the importance of membrane tension in regulating the stability and dynamics of membrane geometry. Recent progresses in understanding the role of membrane tension in regulating dynamical cellular processes are also reviewed. Protein density is emphasized as a key factor in regulating membrane shape transitions: a threshold density of curvature coupling proteins is required for inducing membrane morphology transitions. PMID:24529968

  2. Pathway of membrane fusion with two tension-dependent energy barriers

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2007-01-01

    Fusion of bilayer membranes is studied via dissipative particle dynamics (DPD) simulations. A new set of DPD parameters is introduced which leads to an energy barrier for flips of lipid molecules between adhering membranes. A large number of fusion events is monitored for a vesicle in contact...

  3. Polysaccharide-supported planar bilayer lipid model membranes

    OpenAIRE

    Baumgart, T.; Offenhäusser, A.

    2003-01-01

    Bilayer lipid membranes were deposited onto two different thin water swellable polymer cushions predominantly by Langmuir-Blodgett trough methods. Membranes consisting of zwitterionic lipids supported by agarose films were shown to be unstable, as observed by fluorescence microscopy, reflection interference contrast microscopy, and the impossibility of bilayer spreading (Radler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539-4548) on the agarose surface. Chitosan, formerly observed to pe...

  4. Mechanism of unassisted ion transport across membrane bilayers

    Science.gov (United States)

    Wilson, M. A.; Pohorille, A.

    1996-01-01

    To establish how charged species move from water to the nonpolar membrane interior and to determine the energetic and structural effects accompanying this process, we performed molecular dynamics simulations of the transport of Na+ and Cl- across a lipid bilayer located between two water lamellae. The total length of molecular dynamics trajectories generated for each ion was 10 ns. Our simulations demonstrate that permeation of ions into the membrane is accompanied by the formation of deep, asymmetric thinning defects in the bilayer, whereby polar lipid head groups and water penetrate the nonpolar membrane interior. Once the ion crosses the midplane of the bilayer the deformation "switches sides"; the initial defect slowly relaxes, and a defect forms in the outgoing side of the bilayer. As a result, the ion remains well solvated during the process; the total number of oxygen atoms from water and lipid head groups in the first solvation shell remains constant. A similar membrane deformation is formed when the ion is instantaneously inserted into the interior of the bilayer. The formation of defects considerably lowers the free energy barrier to transfer of the ion across the bilayer and, consequently, increases the permeabilities of the membrane to ions, compared to the rigid, planar structure, by approximately 14 orders of magnitude. Our results have implications for drug delivery using liposomes and peptide insertion into membranes.

  5. Tethered and Polymer Supported Bilayer Lipid Membranes: Structure and Function

    Directory of Open Access Journals (Sweden)

    Jakob Andersson

    2016-05-01

    Full Text Available Solid supported bilayer lipid membranes are model systems to mimic natural cell membranes in order to understand structural and functional properties of such systems. The use of a model system allows for the use of a wide variety of analytical tools including atomic force microscopy, impedance spectroscopy, neutron reflectometry, and surface plasmon resonance spectroscopy. Among the large number of different types of model membranes polymer-supported and tethered lipid bilayers have been shown to be versatile and useful systems. Both systems consist of a lipid bilayer, which is de-coupled from an underlying support by a spacer cushion. Both systems will be reviewed, with an emphasis on the effect that the spacer moiety has on the bilayer properties.

  6. Viscoelastic changes measured in partially suspended single bilayer membranes.

    Science.gov (United States)

    Hasan, Imad Younus; Mechler, Adam

    2015-07-21

    For studies involving biomimetic phospholipid membrane systems, such as membrane-protein interactions, it is crucial that the supported membrane is biomimetic in its physical properties as well as in its composition. Two often overlooked aspects of biomimicry are the need for unrestrained lipid mobility, reflected in the viscoelastic properties of the membrane, and sufficient space between the membrane and the support for the insertion of transmembrane proteins. Here we show for a series of DMPC-based membranes that a partially suspended single bilayer membrane can be formed on functionalized gold surface without tethering. These membranes exhibit sufficient freedom of motion to represent the viscoelastic properties of a free lamellar bilayer membrane as demonstrated by determining the phase transition temperatures of these single bilayer membranes from the viscosity change upon chain melting using the dissipation signal of a quartz crystal microbalance (QCM-D). Atomic force microscopy imaging confirmed confluent, smooth membrane coverage of the QCM-D sensor that completely obscured the roughness of the sputtered gold surface. High-force AFM imaging was able to push membrane patches into the valleys of the gold morphology, confirming the inherently suspended nature of the MPA supported membrane. We show that the correlation between frequency and dissipation changes in the QCM-D sensograms is a sensitive indicator of the morphology of the membrane. PMID:26073288

  7. The lipid bilayer membrane and its interactions with additives.

    NARCIS (Netherlands)

    Meijer, L.A.

    1994-01-01

    The aim of this study was to make accurate predictions on the interaction of biologically relevant molecules with lipid bilayer membranes. We emphasised on the partitioning of these molecules between the membrane phase, and the aqueous phase quantified by the partition coefficient. To make detailed

  8. Shape Changes of Self-Assembled Actin Bilayer Composite Membranes

    CERN Document Server

    Hackl, W; Sackmann, E

    1997-01-01

    We report the self-assembly of thin actin shells beneath the membranes of giant vesicles. Ion-carrier mediated influx of Mg2+ induces actin polymerization in the initially spherical vesicles. Buckling of the vesicles and the formation of blisters after thermally induced bilayer expansion is demonstrated. Bilayer flickering is dominated by tension generated by its coupling to the actin cortex. Quantitative flicker analysis suggests the bilayer and the actin cortex are separated by 0.4 \\mum to 0.5 \\mum due to undulation forces.

  9. Membrane fusion during phage lysis.

    Science.gov (United States)

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

    2015-04-28

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

  10. Equilibrium Configurations of Lipid Bilayer Membranes and Carbon Nanostructures

    Institute of Scientific and Technical Information of China (English)

    Iva(i)lo M.Mladenov; Peter A.Djondjorov; Mariana Ts.Hadzhilazova; Vassil M.Vassilev

    2013-01-01

    The present article concerns the continuum modelling of the mechanical behaviour and equilibrium shapes of two types of nano-scale objects:fluid lipid bilayer membranes and carbon nanostructures.A unified continuum model is used to handle four different case studies.Two of them consist in representing in analytic form cylindrical and axisymmetric equilibrium configurations of single-wall carbon nanotubes and fluid lipid bilayer membranes subjected to uniform hydrostatic pressure.The third one is concerned with determination of possible shapes of junctions between a single-wall carbon nanotube and a fiat graphene sheet or another single-wall carbon nanotube.The last one deals with the mechanical behaviour of closed fluid lipid bilayer membranes (vesicles) adhering onto a fiat homogeneous rigid substrate subjected to micro-injection and uniform hydrostatic pressure.

  11. Physical Aspects of Viral Membrane Fusion

    OpenAIRE

    Laura Wessels; Keith Weninger

    2009-01-01

    Enveloped viruses commonly employ membrane fusion during cell penetration in order to deliver their genetic material across the cell boundary. Large conformational changes in the proteins embedded in the viral membrane play a fundamental role in the membrane fusion process. Despite the tremendously wide variety of viruses that contain membranes, it appears that they all contain membrane fusion protein machinery with a remarkably conserved mechanism of action. Much of our current biochemical u...

  12. Supported lipid bilayer membranes for water purification by reverse osmosis.

    Science.gov (United States)

    Kaufman, Yair; Berman, Amir; Freger, Viatcheslav

    2010-05-18

    Some biological plasma membranes pass water with a permeability and selectivity largely exceeding those of commercial membranes for water desalination using specialized trans-membrane proteins aquaporins. However, highly selective transport of water through aquaporins is usually driven by an osmotic rather mechanical pressure, which is not as attractive from the engineering point of view. The feasibility of adopting biomimetic membranes for water purification driven by a mechanical pressure, i.e., filtration is explored in this paper. Toward this goal, it is proposed to use a commercial nanofiltration (NF) membrane as a support for biomimetic lipid bilayer membranes to render them robust enough to withstand the required pressures. It is shown in this paper for the first time that by properly tuning molecular interactions supported phospholipid bilayers (SPB) can be prepared on a commercial NF membrane. The presence of SPB on the surface was verified and quantified by several spectroscopic and microscopic techniques, which showed morphology close to the desired one with very few defects. As an ultimate test it is shown that hydraulic permeability of the SPB supported on the NF membrane (NTR-7450) approaches the values deduced from the typical osmotic permeabilities of intact continuous bilayers. This permeability was unaffected by the trans-membrane flow of water and by repeatedly releasing and reapplying a 10 bar pressure. Along with a parallel demonstration that aquaporins could be incorporated in a similar bilayer on mica, this demonstrates the feasibility of the proposed approach. The prepared SPB structure may be used as a platform for preparing biomimetic filtration membranes with superior performance based on aquaporins. The concept of SPBs on permeable substrates of the present type may also be useful in the future for studying transport of various molecules through trans-membrane proteins. PMID:20099798

  13. Membrane lysis during biological membrane fusion: collateral damage by misregulated fusion machines

    OpenAIRE

    Engel, Alex; Walter, Peter

    2008-01-01

    In the canonical model of membrane fusion, the integrity of the fusing membranes is never compromised, preserving the identity of fusing compartments. However, recent molecular simulations provided evidence for a pathway to fusion in which holes in the membrane evolve into a fusion pore. Additionally, two biological membrane fusion models—yeast cell mating and in vitro vacuole fusion—have shown that modifying the composition or altering the relative expression levels of membrane fusion comple...

  14. The fusion of membranes and vesicles: pathway and energy barriers from Dissipative Particle Dynamics

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2009-01-01

    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...... 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...

  15. Rabies Virus-Induced Membrane Fusion Pathway

    OpenAIRE

    Gaudin, Yves

    2000-01-01

    Fusion of rabies virus with membranes is triggered at low pH and is mediated by the viral glycoprotein (G). The rabies virus-induced fusion pathway was studied by investigating the effects of exogenous lipids having various dynamic molecular shapes on the fusion process. Inverted cone-shaped lysophosphatidylcholines (LPCs) blocked fusion at a stage subsequent to fusion peptide insertion into the target membrane. Consistent with the stalk-hypothesis, LPC with shorter alkyl chains inhibited fus...

  16. Kinetics of domain registration in multicomponent lipid bilayer membranes

    DEFF Research Database (Denmark)

    Sornbundit, K.; Modchang, C.; Triampo, W.;

    2014-01-01

    The kinetics of registration of lipid domains in the apposing leaflets of symmetric bilayer membranes is investigated via systematic dissipative particle dynamics simulations. The decay of the distance between the centres of mass of the domains in the apposing leaflets is almost linear during early...

  17. Molecular dynamics simulation of melittin in a dimyristoylphosphatidylcholine bilayer membrane.

    OpenAIRE

    Bernèche, S; Nina, M; Roux, B

    1998-01-01

    Molecular dynamics trajectories of melittin in an explicit dimyristoyl phosphatidylcholine (DMPC) bilayer are generated to study the details of lipid-protein interactions at the microscopic level. Melittin, a small amphipathic peptide found in bee venom, is known to have a pronounced effect on the lysis of membranes. The peptide is initially set parallel to the membrane-solution interfacial region in an alpha-helical conformation with unprotonated N-terminus. Solid-state nuclear magnetic reso...

  18. The lipid bilayer membrane and its interactions with additives.

    OpenAIRE

    Meijer, L.A.

    1994-01-01

    The aim of this study was to make accurate predictions on the interaction of biologically relevant molecules with lipid bilayer membranes. We emphasised on the partitioning of these molecules between the membrane phase, and the aqueous phase quantified by the partition coefficient. To make detailed predictions a theory had to be set up along the lines of the self-consistent-field theory developed by Scheutjens and Fleer and extended by Evers, Leermakers, Van Lent, Böhmer, Barneveld, Israëls, ...

  19. Local membrane mechanics of pore-spanning bilayers.

    Science.gov (United States)

    Mey, Ingo; Stephan, Milena; Schmitt, Eva K; Müller, Martin Michael; Ben Amar, Martine; Steinem, Claudia; Janshoff, Andreas

    2009-05-27

    The mechanical behavior of lipid bilayers spanning the pores of highly ordered porous silicon substrates was scrutinized by local indentation experiments as a function of surface functionalization, lipid composition, solvent content, indentation velocity, and pore radius. Solvent-containing nano black lipid membranes (nano-BLMs) as well as solvent-free pore-spanning bilayers were imaged by fluorescence and atomic force microscopy prior to force curve acquisition, which allows distinguishing between membrane-covered and uncovered pores. Force indentation curves on pore-spanning bilayers attached to functionalized hydrophobic porous silicon substrates reveal a predominately linear response that is mainly attributed to prestress in the membranes. This is in agreement with the observation that indentation leads to membrane lysis well below 5% area dilatation. However, membrane bending and lateral tension dominate over prestress and stretching if solvent-free supported membranes obtained from spreading giant liposomes on hydrophilic porous silicon are indented. An elastic regime diagram is presented that readily allows determining the dominant contribution to the mechanical response upon indentation as a function of load and pore radius. PMID:19453196

  20. Lipid Bilayer Membrane Perturbation by Embedded Nanopores: A Simulation Study.

    Science.gov (United States)

    Garcia-Fandiño, Rebeca; Piñeiro, Ángel; Trick, Jemma L; Sansom, Mark S P

    2016-03-22

    A macromolecular nanopore inserted into a membrane may perturb the dynamic organization of the surrounding lipid bilayer. To better understand the nature of such perturbations, we have undertaken a systematic molecular dynamics simulation study of lipid bilayer structure and dynamics around three different classes of nanopore: a carbon nanotube, three related cyclic peptide nanotubes differing in the nature of their external surfaces, and a model of a β-barrel nanopore protein. Periodic spatial distributions of several lipid properties as a function of distance from the nanopore were observed. This was especially clear for the carbon nanotube system, for which the density of lipids, the bilayer thickness, the projection of lipid head-to-tail vectors onto the membrane plane, and lipid lateral diffusion coefficients exhibited undulatory behavior as a function of the distance from the surface of the channel. Overall, the differences in lipid behavior as a function of the nanopore structure reveal local adaptation of the bilayer structure and dynamics to different embedded nanopore structures. Both the local structure and dynamic behavior of lipids around membrane-embedded nanopores are sensitive to the geometry and nature of the outer surface of the macromolecule/molecular assembly forming the pore. PMID:26943498

  1. Bilayer thickness mismatch controls domain size in biomimetic membranes

    Science.gov (United States)

    Heberle, Frederick A.; Petruzielo, Robin S.; Pan, Jianjun; Drazba, Paul; Kučerka, Norbert; Standaert, Robert F.; Feigenson, Gerald W.; Katsara, John

    2013-03-01

    In order to promote functionality, cells may alter the spatial organization of membrane lipids and proteins, including separation of liquid phases into distinct domains. In model membranes, domain size and morphology depend strongly on composition and temperature, but the physicochemical mechanisms controlling them are poorly understood. Theoretical work suggests a role for interfacial energy at domain boundaries, which may be driven in part by thickness mismatch between a domain and its surrounding bilayer. However, no direct evidence linking thickness mismatch to domain size in free-standing bilayers has been reported. We describe the use of Small Angle Neutron Scattering (SANS) to detect domains in simplified lipid-only models that mimic the composition of plasma membrane. We find that domain size is controlled by the degree of acyl chain unsaturation of low-melting temperature lipids, and that this size transition is correlated to changes in the thickness mismatch between coexisting liquid phases.

  2. The mechanoelectrical response of droplet interface bilayer membranes.

    Science.gov (United States)

    Freeman, E C; Najem, J S; Sukharev, S; Philen, M K; Leo, D J

    2016-03-16

    Mechanotransduction and interfacial properties in unsupported liquid biomimetic membranes are explored using the droplet-interface bilayer technique. The fluidic monolayer-membrane system afforded by this technique allows for dynamic control over the membrane dimensions and curvature, which under periodic deformations generates capacitive currents (akin to a Kelvin probe), and permits a detailed electrostatic characterization of the boundary layers as well as observation of flexoelectric effects. Both high and low displacement frequency regimes are examined, and the results show that the mechanoelectric signals generated by the membranes may be linked to the membrane electrostatic structure. In addition, we show that periodic membrane bending in a high-frequency regime generates tension sufficient to activate reconstituted mechanosensitive channels. PMID:26905644

  3. Formation and fluidity measurement of supported lipid bilayer on polyvinyl chloride membrane

    Energy Technology Data Exchange (ETDEWEB)

    Kobayashi, Takuji, E-mail: kobayashi-t@int.ee.tut.ac.jp; Kono, Akiteru, E-mail: kobayashi-t@int.ee.tut.ac.jp; Sawada, Kazuaki [Department of Electrical and Electronic Information Engineering, Toyohashi University of Technology, 1-1 Hibarigaoka Tempaku-cho, Toyohashi, 441-8580 (Japan); Futagawa, Masato [Department of Electrical and Electronic Information Engineering and Head Office for the Tailor-Made and Baton-Zone Graduate Course, Toyohashi University of Technology, 1-1 Hibarigaoka Tempaku-cho, Toyohashi, 441-8580 (Japan); Tero, Ryugo, E-mail: tero@tut.jp [Electronics-Inspired Interdisciplinary Research Institute and Department of Environmental and Life Sciences, Toyohashi University of Technology, 1-1 Hibarigaoka Tempaku-cho, Toyohashi, 441-8580 (Japan)

    2014-02-20

    We prepared an artificial lipid bilayer on a plasticized poly(vinyl chloride) (PVC) membrane on a Si3N4 layer deposited on a Si wafer. We optimized the experimental condition for the fabrication of the PVC membrane, and obtained a PVC membrane with a flat and uniform surface on the scale of several hundreds of micrometer suitable for a substrate for supported lipid bilayers (SLBs). The SLB of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was formed on the PVC membrane by the vesicle fusion method. The observation with a conventional epi-fluorescence microscope and a confocal laser scanning microscope gave geometrically uniform images of the SLB on the PVC membrane. The fluidity and the mobile fraction of the SLB was evaluated by the fluorescence recovery after photobleaching method, and compared with that on a thermally oxidized SiO{sub 2}/Si substrate. The SLB on the PVC membrane contained immobile fraction ∼30%, but the diffusion in the mobile fraction was two times faster than that in the SLB on SiO{sub 2}/Si, which had little immobile fraction.

  4. Fusion Machinery

    DEFF Research Database (Denmark)

    Sørensen, Jakob Balslev; Milosevic, Ira

    2015-01-01

    SNARE proteins constitute the minimal machinery needed for membrane fusion. SNAREs operate by forming a complex, which pulls the lipid bilayers into close contact and provides the mechanical force needed for lipid bilayer fusion. At the chemical synapse, SNARE-complex formation between the vesicu......SNARE proteins constitute the minimal machinery needed for membrane fusion. SNAREs operate by forming a complex, which pulls the lipid bilayers into close contact and provides the mechanical force needed for lipid bilayer fusion. At the chemical synapse, SNARE-complex formation between...

  5. Elastic deformation and failure of lipid bilayer membranes containing cholesterol.

    OpenAIRE

    Needham, D; Nunn, R. S.

    1990-01-01

    Giant bilayer vesicles were reconstituted from several lipids and lipid/cholesterol (CHOL) mixtures: stearolyloleoylphosphatidylcholine (SOPC), bovine sphingomyelin (BSM), diarachidonylphosphatidylcholine (DAPC), SOPC/CHOL, BSM/CHOL, DAPC/CHOL, and extracted red blood cell (RBC) lipids with native cholesterol. Single-walled vesicles were manipulated by micropipette suction and several membrane material properties were determined. The properties measured were the elastic area compressibility m...

  6. Membrane Protein Mobility and Orientation Preserved in Supported Bilayers Created Directly from Cell Plasma Membrane Blebs.

    Science.gov (United States)

    Richards, Mark J; Hsia, Chih-Yun; Singh, Rohit R; Haider, Huma; Kumpf, Julia; Kawate, Toshimitsu; Daniel, Susan

    2016-03-29

    Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins. Fluorescent proteins were tracked, and their diffusion in supported bilayers characterized, using single molecule tracking and moment scaling spectrum (MSS) analysis. Diffusion was characterized for individual proteins as either free or confined, revealing details of the local lipid membrane heterogeneity surrounding the protein. A particularly useful result of our bilayer formation process is the protein orientation in the supported planar bilayer. For both the GPI-linked and transmembrane proteins used here, an enzymatic assay revealed that protein orientation in the planar bilayer results in the extracellular domains facing toward the bulk, and that the dominant mode of bleb rupture is via the "parachute" mechanism. Mobility, orientation, and preservation of the native lipid environment of the proteins using cell blebs offers advantages over proteoliposome reconstitution or disrupted cell membrane preparations, which necessarily result in significant scrambling of protein orientation and typically immobilized membrane proteins in SLBs. The bleb-based bilayer platform presented here is an important step toward integrating membrane proteomic studies on chip, especially for future studies aimed at understanding fundamental effects of lipid interactions

  7. Minimum Membrane Bending Energies of Fusion Pores

    OpenAIRE

    Jackson, Meyer B.

    2009-01-01

    Membranes fuse by forming highly curved intermediates, culminating in structures described as fusion pores. These hourglass-like figures that join two fusing membranes have high bending energies, which can be estimated using continuum elasticity models. Fusion pore bending energies depend strongly on shape, and the present study developed a method for determining the shape that minimizes bending energy. This was first applied to a fusion pore modeled as a single surface and then extended to a...

  8. Effect of the HIV-1 fusion peptide on the mechanical properties and leaflet coupling of lipid bilayers

    Science.gov (United States)

    Shchelokovskyy, P.; Tristram-Nagle, S.; Dimova, R.

    2011-02-01

    The fusion peptide (FP) of the human immunodeficiency virus (HIV) is part of the N-terminus of the viral envelope glycoprotein gp41 and is believed to play an important role in the viral entry process. To understand the immediate effect of this peptide on the cell membrane, we have studied the influence of the synthetic FP sequence FP23 on the mechanical properties of model lipid bilayers. For this purpose, giant unilamellar vesicles were prepared from the unsaturated lipid dioleoylphosphatidylcholine mixed in various molar ratios with FP23. The bending stiffness of the vesicles was measured with two different methods: fluctuation analysis and aspiration with micropipettes. The data obtained from both of these approaches show that the bending stiffness of the membrane decreases gradually with increasing concentration of the FP23 in the bilayer. Low concentrations of only a few mol% FP23 are sufficient to decrease the bending stiffness of the lipid bilayer by about a factor of 2. Finally, data obtained for the stretching elasticity modulus of the membrane suggest that the peptide insertion decreases the coupling between the two leaflets of the bilayer.

  9. Effect of the HIV-1 fusion peptide on the mechanical properties and leaflet coupling of lipid bilayers

    International Nuclear Information System (INIS)

    The fusion peptide (FP) of the human immunodeficiency virus (HIV) is part of the N-terminus of the viral envelope glycoprotein gp41 and is believed to play an important role in the viral entry process. To understand the immediate effect of this peptide on the cell membrane, we have studied the influence of the synthetic FP sequence FP23 on the mechanical properties of model lipid bilayers. For this purpose, giant unilamellar vesicles were prepared from the unsaturated lipid dioleoylphosphatidylcholine mixed in various molar ratios with FP23. The bending stiffness of the vesicles was measured with two different methods: fluctuation analysis and aspiration with micropipettes. The data obtained from both of these approaches show that the bending stiffness of the membrane decreases gradually with increasing concentration of the FP23 in the bilayer. Low concentrations of only a few mol% FP23 are sufficient to decrease the bending stiffness of the lipid bilayer by about a factor of 2. Finally, data obtained for the stretching elasticity modulus of the membrane suggest that the peptide insertion decreases the coupling between the two leaflets of the bilayer.

  10. Melittin-induced cholesterol reorganization in lipid bilayer membranes.

    Science.gov (United States)

    Qian, Shuo; Heller, William T

    2015-10-01

    The peptide melittin, a 26 amino acid, cationic peptide from honey bee (Apis mellifera) venom, disrupts lipid bilayer membranes in a concentration-dependent manner. Rather than interacting with a specific receptor, the peptide interacts directly with the lipid matrix of the membrane in a manner dependent on the lipid composition. Here, a small-angle neutron scattering study of the interaction of melittin with lipid bilayers made of mixtures of dimyristoylphosphatidylcholine (DMPC) and cholesterol (Chol) is presented. Through the use of deuterium-labeled DMPC, changes in the distribution of the lipid and cholesterol in unilamellar vesicles were observed for peptide concentrations below those that cause pores to form. In addition to disrupting the in-plane organization of Chol, melittin produces vesicles having inner and outer leaflet compositions that depend on the lipid-Chol molar ratio and on the peptide concentration. The changes seen at high cholesterol and low peptide concentration are similar to those produced by alamethicin (Qian, S. et al., J. Phys. Chem. B 2014, 118, 11200-11208), which points to an underlying physical mechanism driving the redistribution of Chol, but melittin displays an additional effect not seen with alamethicin. A model for how the peptide drives the redistribution of Chol is proposed. The results suggest that redistribution of the lipids in a target cell membrane by membrane active peptides takes places as a prelude to the lysis of the cell. PMID:26074009

  11. Atomic Force Microscope Studies of the Fusion of Floating Lipid Bilayers

    OpenAIRE

    Abdulreda, Midhat H.; Moy, Vincent T.

    2007-01-01

    This study investigated the fusion of apposing floating bilayers of egg L-α-phosphatidylcholine (egg PC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine. Atomic force microscope measurements of fusion forces under different compression rates were acquired to reveal the energy landscape of the fusion process under varied lipid composition and temperature. Between compression rates of ∼1000 and ∼100,000 pN/s, applied forces in the range from ∼100 to ∼500 pN resulted in fusion of floating bilayer...

  12. Shear-Induced Membrane Fusion in Viscous Solutions

    KAUST Repository

    Kogan, Maxim

    2014-05-06

    Large unilamellar lipid vesicles do not normally fuse under fluid shear stress. They might deform and open pores to relax the tension to which they are exposed, but membrane fusion occurring solely due to shear stress has not yet been reported. We present evidence that shear forces in a viscous solution can induce lipid bilayer fusion. The fusion of 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC) liposomes is observed in Couette flow with shear rates above 3000 s-1 provided that the medium is viscous enough. Liposome samples, prepared at different viscosities using a 0-50 wt % range of sucrose concentration, were studied by dynamic light scattering, lipid fusion assays using Förster resonance energy transfer (FRET), and linear dichroism (LD) spectroscopy. Liposomes in solutions with 40 wt % (or more) sucrose showed lipid fusion under shear forces. These results support the hypothesis that under suitable conditions lipid membranes may fuse in response to mechanical-force- induced stress. © 2014 American Chemical Society.

  13. Surface-Enhanced Infrared Spectroscopy and Neutron Reflectivity Studies of Ubiquinone in Hybrid Bilayer Membranes under Potential Control.

    Science.gov (United States)

    Quirk, Amanda; Lardner, Michael J; Tun, Zin; Burgess, Ian J

    2016-03-01

    Surface-enhanced infrared adsorption spectroscopy (SEIRAS) and neutron reflectometry (NR) were employed to characterize ubiquinone (UQ) containing hybrid bilayer membranes. The biomimetic membrane was prepared by fusing phospholipid vesicles on a hydrophobic octadecanethiol monolayer self-assembled on a thin gold film. Using SEIRAS, the assembly of the membrane is monitored in situ. The presence of ubiquinone is verified by the characteristic carbonyl peaks from the quinone ester. A well-ordered distal lipid leaflet results from fusion of vesicles with and without the addition of ubiquinone. With applied potential, the hybrid bilayer membrane in the absence of UQ behaves in the same way as previously reported solid supported phospholipid membranes. When ubiquinone is incorporated in the membrane, electric field induced changes in the distal leaflet are suppressed. Changes in the infrared vibrations of the ubiquinone due to applied potential indicate the head groups are located in both polar and nonpolar environments. The spectroscopic data reveal that the isoprenoid unit of the ubiquinone is likely lying in the midplane of the lipid bilayer while the head has some freedom to move within the hydrophobic core. The SEIRAS experiments show redox behavior of UQ incorporated in a model lipid membrane that are otherwise inaccessible with traditional electrochemistry techniques. PMID:26867110

  14. Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes

    DEFF Research Database (Denmark)

    Lundbæk, Jens August; Collingwood, S.A.; Ingolfsson, H.I.;

    2010-01-01

    Membrane protein function is regulated by the host lipid bilayer composition. This regulation may depend on specific chemical interactions between proteins and individual molecules in the bilayer, as well as on non-specific interactions between proteins and the bilayer behaving as a physical enti...... use of gramicidin channels as molecular force probes for studying this mechanism, with a unique ability to discriminate between consequences of changes in monolayer curvature and bilayer elastic moduli....

  15. Transport methods for probing the barrier domain of lipid bilayer membranes.

    OpenAIRE

    Xiang, T X; Chen, X.; Anderson, B. D.

    1992-01-01

    Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) ...

  16. Separate fusion of outer and inner mitochondrial membranes

    OpenAIRE

    Malka, Florence; Guillery, Olwenn; Cifuentes-Diaz, Carmen; Guillou, Emmanuelle; Belenguer, Pascale; Lombès, Anne; Rojo, Manuel

    2005-01-01

    Mitochondria are enveloped by two closely apposed boundary membranes with different properties and functions. It is known that they undergo fusion and fission, but it has remained unclear whether outer and inner membranes fuse simultaneously, coordinately or separately. We set up assays for the study of inner and outer membrane fusion in living human cells. Inner membrane fusion was more sensitive than outer membrane fusion to inhibition of glycolysis. Fusion of the inner membrane, but not of...

  17. Automated Lipid Bilayer Membrane Formation Using a Polydimethylsiloxane Thin Film.

    Science.gov (United States)

    Choi, Sangbaek; Yoon, Sunhee; Ryu, Hyunil; Kim, Sun Min; Jeon, Tae-Joon

    2016-01-01

    An artificial lipid bilayer, or black lipid membrane (BLM), is a powerful tool for studying ion channels and protein interactions, as well as for biosensor applications. However, conventional BLM formation techniques have several drawbacks and they often require specific expertise and laborious processes. In particular, conventional BLMs suffer from low formation success rates and inconsistent membrane formation time. Here, we demonstrate a storable and transportable BLM formation system with controlled thinning-out time and enhanced BLM formation rate by replacing conventionally used films (polytetrafluoroethylene, polyoxymethylene, polystyrene) to polydimethylsiloxane (PDMS). In this experiment, a porous-structured polymer such as PDMS thin film is used. In addition, as opposed to conventionally used solvents with low viscosity, the use of squalene permitted a controlled thinning-out time via slow solvent absorption by PDMS, prolonging membrane lifetime. In addition, by using a mixture of squalene and hexadecane, the freezing point of the lipid solution was increased (~16 °C), in addition, membrane precursors were produced that can be indefinitely stored and readily transported. These membrane precursors have reduced BLM formation time of gramicidin A demonstrated the feasibility of the membrane system. PMID:27501114

  18. Millimeter microwave effect on ion transport across lipid bilayer membranes

    Energy Technology Data Exchange (ETDEWEB)

    Alekseev, S.I. [Russian Academy of Sciences, Pushchino (Russian Federation). Inst. of Cell Biophysics; Ziskin, M.C. [Temple Univ. Medical School, Philadelphia, PA (United States). Center for Biomedical Physics

    1995-06-01

    The effects of millimeter microwaves in the frequency range of 54--76 GHz on capacitance and conductance of lipid bilayer membranes (BLM) were studied. Some of the membranes were modified by gramicidin A and amphotericin B or by tetraphenylboron anions (TPhB{sup {minus}}). The millimeter microwaves were pulse-modulated (PW) at repetition rates ranging from 1 to 100 pps, PW at 1,000 pps, or unmodulated continuous waves (CW). The maximum output power at the waveguide outlet was 20 mW. It was found that CW irradiation decreased the unmodified BLM capacitance by 1.2% {+-} 0.5%. At the same time, membrane current induced by TPhB{sup {minus}} transport increased by 5% {+-} 1%. The changes in conductance of ionic channels formed by gramicidin A and amphotericin B were small (0.6% {+-} 0.4%). No resonance-like effects of mm-wave irradiation on membrane capacitance, ionic channel currents, or TPhB{sup {minus}} transport were detected. All changes in membrane capacitance and currents were independent of the modulation employed and were equivalent to heating by approximately 1.1 C.

  19. Multiscale modeling of droplet interface bilayer membrane networks.

    Science.gov (United States)

    Freeman, Eric C; Farimani, Amir B; Aluru, Narayana R; Philen, Michael K

    2015-11-01

    Droplet interface bilayer (DIB) networks are considered for the development of stimuli-responsive membrane-based materials inspired by cellular mechanics. These DIB networks are often modeled as combinations of electrical circuit analogues, creating complex networks of capacitors and resistors that mimic the biomolecular structures. These empirical models are capable of replicating data from electrophysiology experiments, but these models do not accurately capture the underlying physical phenomena and consequently do not allow for simulations of material functionalities beyond the voltage-clamp or current-clamp conditions. The work presented here provides a more robust description of DIB network behavior through the development of a hierarchical multiscale model, recognizing that the macroscopic network properties are functions of their underlying molecular structure. The result of this research is a modeling methodology based on controlled exchanges across the interfaces of neighboring droplets. This methodology is validated against experimental data, and an extension case is provided to demonstrate possible future applications of droplet interface bilayer networks. PMID:26594262

  20. The structural dynamics of the flavivirus fusion peptide-membrane interaction.

    Directory of Open Access Journals (Sweden)

    Ygara S Mendes

    Full Text Available Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP. Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus-cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA(G ((98DRGWGNGCGLFGK(110 and FLA(H ((98DRGWGNHCGLFGK(110, and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA(G and FLA(H were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.

  1. Kinetics of Domains Registration in Multicomponent Lipid Bilayer Membranes

    Science.gov (United States)

    Sornbundit, Kan; Modchang, Charin; Triampo, Wannapong; Triampo, Darapond; Nuttavut, Narin; Sunil Kumar, P.B; Laradji, Mohamed

    2014-01-01

    The kinetics of registration of lipid domains in the apposing leaflets of symmetric bilayer membranes is investigated via systematic dissipative particle dynamics simulations. The decay of the distance between the centres of mass of the domains in the apposing leaflets is almost linear during early stages, and then becomes exponential during late times. The time scales of both linear and exponential decays are found to increase with decreasing the strength of interleaflet coupling. The ratio between the time scales of the exponential and linear regimes decreases with increasing the domain size, implying that the decay of the distance between the domains centres of mass is essentially linear for large domains. These numerical results are largely in agreement with the recent theoretical predictions of Han and Haataja [Soft Matter (2013) 9:2120-2124]. We also found that the domains become elongated during the registration process. PMID:25090030

  2. Effects of Fatty Acid Inclusion in a DMPC Bilayer Membrane

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Hansen, Flemming Yssing; Møller, Martin S.;

    2009-01-01

    packing and structure of oleic acid (HOA) and stearic acid (HSA) in fluid bilayers of dimyristoylphosphatidylcholine (DMPC). The experimental data show a small but consistent positive excess volume for fatty acid concentrations below 10 mol %. At higher concentrations the fatty acids mix ideally with......Free fatty acids in biomembranes have been proposed to be a central component in several cellular control and regulatory mechanisms. To elucidate some fundamental elements underlying this, we have applied molecular dynamics simulations and experimental density measurements to study the molecular...... fluid DMPC. The simulations, which were benchmarked against the densitometric data, revealed interesting differences in the structure and location of the fatty acids depending on their protonation status. Thus, the protonated (uncharged) acid is located rather deeply in the membrane with an average...

  3. Proteolytic cleavage of Opa1 stimulates mitochondrial inner membrane fusion and couples fusion to oxidative phosphorylation

    OpenAIRE

    Mishra, Prashant; Carelli, Valerio; Manfredi, Giovanni; Chan, David C.

    2014-01-01

    Mitochondrial fusion is essential for maintenance of mitochondrial function. The mitofusin GTPases control mitochondrial outer membrane fusion, whereas the dynamin-related GTPase Opa1 mediates inner membrane fusion. We show that mitochondrial inner membrane fusion is tuned by the level of oxidative phosphorylation (OXPHOS), whereas outer membrane fusion is insensitive. Consequently, cells from patients with pathogenic mtDNA mutations show a selective defect in mitochondrial inner membrane fus...

  4. Pressure effects on the equilibrium configurations of bilayer lipid membranes

    Science.gov (United States)

    DeVita, Raffaella; Stewart, Iain W.; Leo, Donald J.

    2007-10-01

    Planar bilayer lipid membranes (BLMs) are currently employed to construct many bio-inspired material systems and structures. In order to characterize the pressure effects on the equilibrium configurations of these biological membranes, a novel continuum model is proposed. The BLM is assumed to be a two-layer smectic A liquid crystal. The mean orientation of the amphiphilic molecules comprising the membrane is postulated to be perpendicular to the layers and each layer is idealized as a two-dimensional liquid. Moreover, the BLM is modeled as a simply supported plate undergoing small deformations. It is subjected to a pressure load that acts perpendicularly to the layers. The equilibrium equations and boundary conditions are derived from the bulk elastic energy for smectic A liquid crystals as described by de Gennes and Prost (1993 The Physics of Liquid Crystals 2nd edn (Oxford Science Publications)) by using variational methods. The resulting fourth-order linear partial differential equation is solved by employing cylindrical functions and the series solution is proved to be convergent. The solution is numerically computed for values of the model parameters that are reported in the literature. This paper is dedicated to the memory of our colleagues, Professors Kevin P Granata and Liviu Librescv, who lost their lives during the sensless tragedy on 16 April, 2007 at Virginia Tech.

  5. Palaeontological evidence of membrane relationship in step-by-step membrane fusion

    OpenAIRE

    Wang, Xin; Liu, Wenzhe; DU, KAIHE

    2010-01-01

    Studies on membrane fusion in living cells indicate that initiation of membrane fusion is a transient and hard to capture process. Despite previous research, membrane behaviour at this point is still poorly understood. Recent palaeobotanical research has revealed snapshots of membrane fusion in a 15-million-year-old fossil pinaceous cone. To reveal the membrane behaviour during the fusion, we conducted more observations on the same fossil material. Several discernible steps of membrane fusion...

  6. Statistical thermodynamics of association colloids : the equilibrium structure of micelles, vesicles and bilayer membranes.

    OpenAIRE

    Leermakers, F.A.M.

    1988-01-01

    The aim of the present study was to unravel the general equilibrium physical properties of lipid bilayer membranes. We consider four major questions:1. What determines the morphology of the association colloids (micelles, membranes, vesicles) in general?2. Do the apolar tails of the lipids in the bilayer organise themselves more like matches in a box or rather like hot spaghetti in a pan?3. How does this membrane organisation depend on temperature?4. How do additives like surfactants or polym...

  7. Nuclear Magnetic Resonance Structural Studies of Membrane Proteins in Micelles and Bilayers

    OpenAIRE

    Gong, Xiao-Min; Franzin, Carla M.; Thai, Khang; Yu, Jinghua; Marassi, Francesca M.

    2007-01-01

    Nuclear magnetic resonance (NMR) spectroscopy enables determination of membrane protein structures in lipid environments, such as micelles and bilayers. This chapter outlines the steps for membrane-protein structure determination using solution NMR with micelle samples, and solid-state NMR with oriented lipid-bilayer samples. The methods for protein expression and purification, sample preparation, and NMR experiments are described and illustrated with examples from γ and CHIF, two membrane pr...

  8. Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

    OpenAIRE

    Marassi, Francesca M.; Crowell, Kevin J.

    2003-01-01

    The preparation of oriented, hydration-optimized lipid bilayer samples, for NMR structure determination of membrane proteins, is described. The samples consist of planar phospholipid bilayers, containing membrane proteins, that are oriented on single pairs of glass slides, and are placed in the coil of the NMR probe with the bilayer plane perpendicular to the direction of the magnetic field. Lipid bilayers provide a medium that closely resembles the biological membrane, and sample orientation...

  9. Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion

    OpenAIRE

    Klupp, Barbara G.; Nixdorf, Ralf; Mettenleiter, Thomas C.

    2000-01-01

    A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as...

  10. A nanohybrid membrane with lipid bilayer-like properties utilized as a conductimetric saccharin sensor.

    Science.gov (United States)

    Chalkias, Nikolaos G; Giannelis, Emmanuel P

    2007-10-31

    Since their introduction, artificial lipid bilayer membranes were used in a wide array of applications, such as sensors, biocompatible materials and study-models of the cell's outer boundary. Here, we present a nanohybrid membrane using an inorganic host and amphiphilic organic molecules with lipid bilayer-like properties. The stability of the presented mimetic membrane is significantly improved when compared to existing methods. The nanohybrid membrane exhibited two thermotropic phases corresponding to the L(alpha) and L(beta) phases that lipid bilayer membranes are known to adopt. Integration of cholesterol molecules into the nanohybrid membrane lead to the same qualitative effects as in lipid bilayers, including expansion of the bilayer spacing and decrease of the L(alpha) to L(beta) transition enthalpy. To further illustrate the similarities of the synthesized membrane with a lipid bilayer, the ability of the nanohybrid membrane to function as saccharin conductimetric sensor was evaluated. The lower limit of detection of the sensor was 6 microM and the linear range of response was from 20 to 400 microM. PMID:17548189

  11. Dissipative Particle Dynamics of Tension-induced Membrane Fusion

    OpenAIRE

    Grafmueller, Andrea; Lipowsky, Reinhard; Shillcock, Julian C.

    2009-01-01

    Abstract Recent studies of tension-induced membrane fusion using dissipative particle dynamics (DPD) simulations are briefly reviewed. The stochastic nature of the fusion process makes it necessary to simulate a large number of fusion attempts in order to obtain reliable fusion statistics and to extract meaningful values for the fusion probability and the average fusion times. All successful fusion events follow the same pathway. In this fusion pathway, configurations of individual...

  12. Prevention of Intra-Abdominal Adhesion by Bi-Layer Electrospun Membrane

    OpenAIRE

    Cunyi Fan; Hede Yan; Wei Wang; Shichao Jiang

    2013-01-01

    The aim of this study was to compare the anti-adhesion efficacy of a bi-layer electrospun fibrous membrane consisting of hyaluronic acid-loaded poly(ε-caprolactone) (PCL) fibrous membrane as the inner layer and PCL fibrous membrane as the outer layer with a single-layer PCL electrospun fibrous membrane in a rat cecum abrasion model. The rat model utilized a cecal abrasion and abdominal wall insult surgical protocol. The bi-layer and PCL membranes were applied between the cecum and the abdomin...

  13. Biodegradable, Tethered Lipid Bilayer-Microsphere Systems with Membrane-Integrated α-Helical Peptide Anchors.

    Science.gov (United States)

    Fried, Eric S; Luchan, Joshua; Gilchrist, M Lane

    2016-04-12

    Supported lipid bilayers (SLBs) are ideally suited for the study of biomembrane-biomembrane interactions and for the biomimicry of cell-to-cell communication, allowing for surface ligand displays that contain laterally mobile elements. However, the SLB paradigm does not include three-dimensionality and biocompatibility. As a way to bypass these limitations, we have developed a biodegradable form of microsphere SLBs, also known as proteolipobeads (PLBs), using PLGA microspheres. Microspheres were synthesized using solvent evaporation and size selected with fluorescence activated cell sorting (FACS). Biomembranes were covalently tethered upon fusion to microsphere supports via short-chain PEG spacers connecting membrane-integrated α-helical peptides and the microsphere surface, affecting membrane diffusivity and mobility as indicated by confocal FRAP analysis. Membrane heterogeneities, which are attributed to PLGA hydrophobicity and rough surface topography, are curtailed by the addition of PEG tethers. This method allows for the presentation of tethered, laterally mobile biomembranes in three dimensions with functionally embedded attachment peptides for mobile ligand displays. PMID:26972467

  14. The Bilayer Enhances Rhodopsin Kinetic Stability in Bovine Rod Outer Segment Disk Membranes

    OpenAIRE

    Corley, Scott C.; Sprangers, Peter; Albert, Arlene D.

    2011-01-01

    Rhodopsin is a kinetically stable protein constituting >90% of rod outer segment disk membrane protein. To investigate the bilayer contribution to rhodopsin kinetic stability, disk membranes were systematically disrupted by octyl-β-D-glucopyranoside. Rhodopsin kinetic stability was examined under subsolubilizing (rhodopsin in a bilayer environment perturbed by octyl-β-D-glucopyranoside) and under fully solubilizing conditions (rhodopsin in a micelle with cosolubilized phospholipids). As deter...

  15. Filament networks attached to membranes: cytoskeletal pressure and local bilayer deformation

    Energy Technology Data Exchange (ETDEWEB)

    Auth, Thorsten [Department of Materials and Interfaces, Weizmann Institute of Science, PO Box 26, Rehovot 76100 (Israel); Safran, S A [Department of Materials and Interfaces, Weizmann Institute of Science, PO Box 26, Rehovot 76100 (Israel); Gov, Nir S [Department of Chemical Physics, Weizmann Institute of Science, PO Box 26, Rehovot 76100 (Israel)

    2007-11-15

    Several cell types, among them red blood cells, have a cortical, two-dimensional (2D) network of filaments sparsely attached to their lipid bilayer. In many mammalian cells, this 2D polymer network is connected to an underlying 3D, more rigid cytoskeleton. In this paper, we consider the pressure exerted by the thermally fluctuating, cortical network of filaments on the bilayer and predict the bilayer deformations that are induced by this pressure. We treat the filaments as flexible polymers and calculate the pressure that a network of such linear chains exerts on the bilayer; we then minimize the bilayer shape in order to predict the resulting local deformations. We compare our predictions with membrane deformations observed in electron micrographs of red blood cells. The polymer pressure along with the resulting membrane deformation can lead to compartmentalization, regulate in-plane diffusion and may influence protein sorting as well as transmit signals to the polymerization of the underlying 3D cytoskeleton.

  16. Fluctuation and dynamics of a lipid bilayer membrane under an electric field

    Science.gov (United States)

    Young, Yuan-Nan; Miksis, Michael; Vlahovska, Petia

    2015-11-01

    Membrane fluctuation and dynamics under an electric field is investigated, and results show that the membrane instability and dynamics depend not only on the mismatch in conductivity and permittivity between the bulk fluids, but also on the membrane charging time. In addition, the (entropic) membrane tension is found to depend on the electric field. Lubrication theory is utilized to examine the nonlinear dynamics of a planar lipid bilayer membrane with and without electrokinetics. Partial support from NSF/DMS 1222550, 1412789.

  17. Regulation of membrane protein function by lipid bilayer elasticity—a single molecule technology to measure the bilayer properties experienced by an embedded protein

    DEFF Research Database (Denmark)

    Lundbæk, Jens August

    2008-01-01

    protein and the host lipid bilayer provide an energetic coupling, whereby protein function can be regulated by the bilayer elasticity. The feasibility of this ‘hydrophobic coupling mechanism’ has been demonstrated using the gramicidin channel, a model membrane protein, in planar lipid bilayers. Using...... voltage-dependent sodium channels, N-type calcium channels and GABAA receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure...

  18. SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy.

    Science.gov (United States)

    Nikolaus, Joerg; Karatekin, Erdem

    2016-01-01

    In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. PMID:27585113

  19. Computational Design of Multi-component Bio-Inspired Bilayer Membranes

    Directory of Open Access Journals (Sweden)

    Evan Koufos

    2014-04-01

    Full Text Available Our investigation is motivated by the need to design bilayer membranes with tunable interfacial and mechanical properties for use in a range of applications, such as targeted drug delivery, sensing and imaging. We draw inspiration from biological cell membranes and focus on their principal constituents. In this paper, we present our results on the role of molecular architecture on the interfacial, structural and dynamical properties of bio-inspired membranes. We focus on four lipid architectures with variations in the head group shape and the hydrocarbon tail length. Each lipid species is composed of a hydrophilic head group and two hydrophobic tails. In addition, we study a model of the Cholesterol molecule to understand the interfacial properties of a bilayer membrane composed of rigid, single-tail molecular species. We demonstrate the properties of the bilayer membranes to be determined by the molecular architecture and rigidity of the constituent species. Finally, we demonstrate the formation of a stable mixed bilayer membrane composed of Cholesterol and one of the phospholipid species. Our approach can be adopted to design multi-component bilayer membranes with tunable interfacial and mechanical properties. We use a Molecular Dynamics-based mesoscopic simulation technique called Dissipative Particle Dynamics that resolves the molecular details of the components through soft-sphere coarse-grained models and reproduces the hydrodynamic behavior of the system over extended time scales.

  20. Effects of Anesthetic Membrane Solutes on Orientational Order in Lecithin Bilayer Membranes: a Deuterium NMR Study.

    Science.gov (United States)

    Phonphok, Nason

    _{z^ ' z^'}.. A theory for the molecular mechanism of anesthesia is proposed. Our working hypothesis is that general anesthetics interact with phosphatidylcholine bilayers in the headgroup region, changing its time-averaged conformation and altering the extent of cation binding to the surface of the membrane. In this manner the electrical properties of the membrane are changed. (Abstract shortened with permission of author.).

  1. Nuclear magnetic resonance structural studies of membrane proteins in micelles and bilayers.

    Science.gov (United States)

    Gong, Xiao-Min; Franzin, Carla M; Thai, Khang; Yu, Jinghua; Marassi, Francesca M

    2007-01-01

    Nuclear magnetic resonance (NMR) spectroscopy enables determination of membrane protein structures in lipid environments, such as micelles and bilayers. This chapter outlines the steps for membrane-protein structure determination using solution NMR with micelle samples, and solid-state NMR with oriented lipid-bilayer samples. The methods for protein expression and purification, sample preparation, and NMR experiments are described and illustrated with examples from gamma and CHIF, two membrane proteins that function as regulatory subunits of the Na+- and K+-ATPase. PMID:17951757

  2. Elastic properties and line tension of self-assembled bilayer membranes

    Science.gov (United States)

    Li, Jianfeng; Pastor, Kyle A.; Shi, An-Chang; Schmid, Friederike; Zhou, Jiajia

    2013-07-01

    The elastic properties of a self-assembled bilayer membrane are studied using the self-consistent field theory, applied to a model system composed of flexible amphiphilic chains dissolved in hydrophilic polymeric solvents. Examining the free energy of bilayer membranes with different geometries allows us to calculate their bending modulus, Gaussian modulus, two fourth-order membrane moduli, and the line tension. The dependence of these parameters on the microscopic characteristics of the amphiphilic chain, characterized by the volume fraction of the hydrophilic component, is systematically studied. The theoretical predictions are compared with the results from a simple monolayer model, which approximates a bilayer membrane by two monolayers. The region of validity of the linear elasticity theory is analyzed by examining the higher-order contributions.

  3. Fusion of biomimetic stealth probes into lipid bilayer cores

    OpenAIRE

    Almquist, Benjamin D.; Melosh, Nicholas A.

    2010-01-01

    Many biomaterials are designed to regulate the interactions between artificial and natural surfaces. However, when materials are inserted through the cell membrane itself the interface formed between the interior edge of the membrane and the material surface is not well understood and poorly controlled. Here we demonstrate that by replicating the nanometer-scale hydrophilic-hydrophobic-hydrophilic architecture of transmembrane proteins, artificial “stealth” probes spontaneously insert and anc...

  4. Photopolymerization of diacetylene lipid bilayers and its application to the construction of micropatterned biomimetic membranes

    OpenAIRE

    Morigaki, K.; Baumgart, T.; Jonas, U.; Offenhäusser, A.

    2002-01-01

    Photopolymerization of diacetylene-containing amphiphiles in substrate-supported bilayers has been studied in connection with the development of a new fabrication strategy of micropatterned biomimetic membrane systems. Two types of amphiphilic diacetylene molecules were compared, one being a monoalkyl phosphate, phosphoric acid monohexacosa-10,12-diynyl ester (1), and the other being a phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (2). The bilayers of monomeric diace...

  5. Theoretical analysis of hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

    OpenAIRE

    Harroun, T A; Heller, W T; Weiss, T M; Yang, L; Huang, H W

    1999-01-01

    We present a quantitative analysis of the effects of hydrophobic matching and membrane-mediated protein-protein interactions exhibited by gramicidin embedded in dimyristoylphosphatidylcholine (DMPC) and dilauroylphosphatidylcholine (DLPC) bilayers (Harroun et al., 1999. Biophys. J. 76:937-945). Incorporating gramicidin, at 1:10 peptide/lipid molar ratio, decreases the phosphate-to-phosphate (PtP) peak separation in the DMPC bilayer from 35.3 A without gramicidin to 32.7 A. In contrast, the sa...

  6. IM30 triggers membrane fusion in cyanobacteria and chloroplasts.

    Science.gov (United States)

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

    2015-01-01

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

  7. Interaction of nuclease colicins with membranes: insertion depth correlates with bilayer perturbation.

    Directory of Open Access Journals (Sweden)

    Mireille Vankemmelbeke

    Full Text Available BACKGROUND: Protein transport across cellular membranes is an important aspect of toxin biology. Escherichia coli cell killing by nuclease colicins occurs through DNA (DNases or RNA (RNases hydrolysis and to this end their cytotoxic domains require transportation across two sets of membranes. In order to begin to unravel the molecular mechanisms underlying the membrane translocation of colicin nuclease domains, we have analysed the membrane association of four DNase domains (E9, a charge reduction E9 mutant, E8, and E7 and one ribosomal RNase domain (E3 using a biomembrane model system. PRINCIPAL RESULTS: We demonstrate, through the use of large unilamellar vesicles composed of synthetic and E. coli lipids and a membrane surface potential sensor, that the colicin nuclease domains bind anionic membranes only, with micromolar affinity and via a cooperative binding mechanism. The evaluation of the nuclease bilayer insertion depth, through a fluorescence quenching analysis using brominated lipids, indicates that the nucleases locate to differential regions in the bilayer. Colicin DNases target the interfacial region of the lipid bilayer, with the DNase E7 showing the deepest insertion, whereas the ribosomal RNase E3 penetrates into the hydrophobic core region of the bilayer. Furthermore, the membrane association of the DNase E7 and the ribosomal RNase E3 induces vesicle aggregation, lipid mixing and content leakage to a much larger extent than that of the other DNases analysed. CONCLUSIONS/SIGNIFICANCE: Our results show, for the first time, that after the initial electrostatically driven membrane association, the pleiotropic membrane effects induced by colicin nuclease domains relate to their bilayer insertion depth and may be linked to their in vivo membrane translocation.

  8. Dissipative Particle Dynamics of tension-induced membrane fusion

    DEFF Research Database (Denmark)

    Shillcock, Julian C.

    2009-01-01

    Recent studies of tension-induced membrane fusion using dissipative particle dynamics (DPD) simulations are briefly reviewed. The stochastic nature of the fusion process makes it necessary to simulate a large number of fusion attempts in order to obtain reliable fusion statistics and to extract...

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

    OpenAIRE

    Risselada, H.; Bubnis, G.; Grubmüller, H.

    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 th...

  10. A Bilayered Structure Comprised of Functionalized Carbon Nanotubes for Desalination by Membrane Distillation.

    Science.gov (United States)

    Bhadra, Madhuleena; Roy, Sagar; Mitra, Somenath

    2016-08-01

    The development of a novel carbon nanotube (CNT) immobilized membrane comprised of a double-layer structure is presented for water desalination by membrane distillation. The bilayered structure is comprised of CNTs functionalized with a hydrophobic octadecyl amine group on the feed side and carboxylated CNTs on the permeate side. The latter is more hydrophilic. The hydrophobic CNTs provide higher water vapor permeation, while the hydrophilic CNTs facilitate the condensation of water vapor. Together, these led to superior performance, and flux in a direct contact membrane distillation mode was found to be as high as 121 kg/m(2)h at 80 °C. The bilayered membrane represented an enhancement of 70% over the unmodified membrane and 37% over a membrane which had a monolayered structure where only the feed side was CNT-modified. PMID:27387851

  11. Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

    OpenAIRE

    Tunuguntla, Ramya; Bangar, Mangesh; Kim, Kyunghoon; Stroeve, Pieter; Ajo-Franklin, Caroline M.; Noy, Aleksandr

    2013-01-01

    Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role o...

  12. Effects of Lipid Composition on Bilayer Membranes Quantified by All-Atom Molecular Dynamics.

    Science.gov (United States)

    Ding, Wei; Palaiokostas, Michail; Wang, Wen; Orsi, Mario

    2015-12-10

    Biological bilayer membranes typically contain varying amounts of lamellar and nonlamellar lipids. Lamellar lipids, such as dioleoylphosphatidylcholine (DOPC), are defined by their tendency to form the lamellar phase, ubiquitous in biology. Nonlamellar lipids, such as dioleoylphosphatidylethanolamine (DOPE), prefer instead to form nonlamellar phases, which are mostly nonbiological. However, nonlamellar lipids mix with lamellar lipids in biomembrane structures that remain overall lamellar. Importantly, changes in the lamellar vs nonlamellar lipid composition are believed to affect membrane function and modulate membrane proteins. In this work, we employ atomistic molecular dynamics simulations to quantify how a range of bilayer properties are altered by variations in the lamellar vs nonlamellar lipid composition. Specifically, we simulate five DOPC/DOPE bilayers at mixing ratios of 1/0, 3/1, 1/1, 1/3, and 0/1. We examine properties including lipid area and bilayer thickness, as well as the transmembrane profiles of electron density, lateral pressure, electric field, and dipole potential. While the bilayer structure is only marginally altered by lipid composition changes, dramatic effects are observed for the lateral pressure, electric field, and dipole potential profiles. Possible implications for membrane function are discussed. PMID:26560961

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

    OpenAIRE

    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...

  14. Strong influence of periodic boundary conditions on lateral diffusion in lipid bilayer membranes

    Science.gov (United States)

    Camley, Brian A.; Lerner, Michael G.; Pastor, Richard W.; Brown, Frank L. H.

    2015-12-01

    The Saffman-Delbrück hydrodynamic model for lipid-bilayer membranes is modified to account for the periodic boundary conditions commonly imposed in molecular simulations. Predicted lateral diffusion coefficients for membrane-embedded solid bodies are sensitive to box shape and converge slowly to the limit of infinite box size, raising serious doubts for the prospects of using detailed simulations to accurately predict membrane-protein diffusivities and related transport properties. Estimates for the relative error associated with periodic boundary artifacts are 50% and higher for fully atomistic models in currently feasible simulation boxes. MARTINI simulations of LacY membrane protein diffusion and LacY dimer diffusion in DPPC membranes and lipid diffusion in pure DPPC bilayers support the underlying hydrodynamic model.

  15. Strong influence of periodic boundary conditions on lateral diffusion in lipid bilayer membranes

    International Nuclear Information System (INIS)

    The Saffman-Delbrück hydrodynamic model for lipid-bilayer membranes is modified to account for the periodic boundary conditions commonly imposed in molecular simulations. Predicted lateral diffusion coefficients for membrane-embedded solid bodies are sensitive to box shape and converge slowly to the limit of infinite box size, raising serious doubts for the prospects of using detailed simulations to accurately predict membrane-protein diffusivities and related transport properties. Estimates for the relative error associated with periodic boundary artifacts are 50% and higher for fully atomistic models in currently feasible simulation boxes. MARTINI simulations of LacY membrane protein diffusion and LacY dimer diffusion in DPPC membranes and lipid diffusion in pure DPPC bilayers support the underlying hydrodynamic model

  16. Strong influence of periodic boundary conditions on lateral diffusion in lipid bilayer membranes

    Energy Technology Data Exchange (ETDEWEB)

    Camley, Brian A. [Center for Theoretical Biological Physics and Department of Physics, University of California, San Diego, California 92093 (United States); Department of Physics, University of California, Santa Barbara, California 93106 (United States); Lerner, Michael G. [Department of Physics and Astronomy, Earlham College, Richmond, Indiana 47374 (United States); Laboratory of Computational Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 (United States); Pastor, Richard W. [Laboratory of Computational Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892 (United States); Brown, Frank L. H. [Department of Physics, University of California, Santa Barbara, California 93106 (United States); Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106 (United States)

    2015-12-28

    The Saffman-Delbrück hydrodynamic model for lipid-bilayer membranes is modified to account for the periodic boundary conditions commonly imposed in molecular simulations. Predicted lateral diffusion coefficients for membrane-embedded solid bodies are sensitive to box shape and converge slowly to the limit of infinite box size, raising serious doubts for the prospects of using detailed simulations to accurately predict membrane-protein diffusivities and related transport properties. Estimates for the relative error associated with periodic boundary artifacts are 50% and higher for fully atomistic models in currently feasible simulation boxes. MARTINI simulations of LacY membrane protein diffusion and LacY dimer diffusion in DPPC membranes and lipid diffusion in pure DPPC bilayers support the underlying hydrodynamic model.

  17. Lipid clustering correlates with membrane curvature as revealed by molecular simulations of complex lipid bilayers.

    Directory of Open Access Journals (Sweden)

    Heidi Koldsø

    2014-10-01

    Full Text Available Cell membranes are complex multicomponent systems, which are highly heterogeneous in the lipid distribution and composition. To date, most molecular simulations have focussed on relatively simple lipid compositions, helping to inform our understanding of in vitro experimental studies. Here we describe on simulations of complex asymmetric plasma membrane model, which contains seven different lipids species including the glycolipid GM3 in the outer leaflet and the anionic lipid, phosphatidylinositol 4,5-bisphophate (PIP2, in the inner leaflet. Plasma membrane models consisting of 1500 lipids and resembling the in vivo composition were constructed and simulations were run for 5 µs. In these simulations the most striking feature was the formation of nano-clusters of GM3 within the outer leaflet. In simulations of protein interactions within a plasma membrane model, GM3, PIP2, and cholesterol all formed favorable interactions with the model α-helical protein. A larger scale simulation of a model plasma membrane containing 6000 lipid molecules revealed correlations between curvature of the bilayer surface and clustering of lipid molecules. In particular, the concave (when viewed from the extracellular side regions of the bilayer surface were locally enriched in GM3. In summary, these simulations explore the nanoscale dynamics of model bilayers which mimic the in vivo lipid composition of mammalian plasma membranes, revealing emergent nanoscale membrane organization which may be coupled both to fluctuations in local membrane geometry and to interactions with proteins.

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

    OpenAIRE

    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 rela...

  19. Regulation of membrane protein function by lipid bilayer elasticity-a single molecule technology to measure the bilayer properties experienced by an embedded protein

    Energy Technology Data Exchange (ETDEWEB)

    Lundbaek, Jens August [Department of Physiology and Biophysics, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021 (United States)

    2006-07-19

    Membrane protein function is generally regulated by the molecular composition of the host lipid bilayer. The underlying mechanisms have long remained enigmatic. Some cases involve specific molecular interactions, but very often lipids and other amphiphiles, which are adsorbed to lipid bilayers, regulate a number of structurally unrelated proteins in an apparently non-specific manner. It is well known that changes in the physical properties of a lipid bilayer (e.g., thickness or monolayer spontaneous curvature) can affect the function of an embedded protein. However, the role of such changes, in the general regulation of membrane protein function, is unclear. This is to a large extent due to lack of a generally accepted framework in which to understand the many observations. The present review summarizes studies which have demonstrated that the hydrophobic interactions between a membrane protein and the host lipid bilayer provide an energetic coupling, whereby protein function can be regulated by the bilayer elasticity. The feasibility of this 'hydrophobic coupling mechanism' has been demonstrated using the gramicidin channel, a model membrane protein, in planar lipid bilayers. Using voltage-dependent sodium channels, N-type calcium channels and GABA{sub A} receptors, it has been shown that membrane protein function in living cells can be regulated by amphiphile induced changes in bilayer elasticity. Using the gramicidin channel as a molecular force transducer, a nanotechnology to measure the elastic properties experienced by an embedded protein has been developed. A theoretical and technological framework, to study the regulation of membrane protein function by lipid bilayer elasticity, has been established.

  20. Evidence for the “Cold Fusion” of Bilayer Membranes

    OpenAIRE

    Ravoo, Bart Jan; Kevelam, Jan; Weringa, Wilke D.; Engberts, Jan B.F.N.

    1998-01-01

    Polymerization of the lipid headgroups inhibits calcium-induced fusion of small unilamellar vesicles of the lipid di-n-dodecyloxypropyl beta-nitrostyryl phosphate but does not influence vesicle aggregation. Addition of a copolymer of lauryl methacrylate and acrylamide (LMPAM) provides the vesicles with a steric shield that prevents both fusion and aggregation. Accurate microcalorimetric determination of the enthalpies of vesicle aggregation and fusion was possible by comparison of titrations ...

  1. Membrane fusion machines of paramyxoviruses: capture of intermediates of fusion

    OpenAIRE

    Charles J Russell; Theodore S Jardetzky; Lamb, Robert A.

    2001-01-01

    Peptides derived from heptad repeat regions adjacent to the fusion peptide and transmembrane domains of many viral fusion proteins form stable helical bundles and inhibit fusion specifically. Paramyxovirus SV5 fusion (F) protein-mediated fusion and its inhibition by the peptides N-1 and C-1 were analyzed. The temperature dependence of fusion by F suggests that thermal energy, destabilizing proline residues and receptor binding by the hemagglutinin–neuraminidase (HN) protein collectively contr...

  2. Electric field increases the phase transition temperature in the bilayer membrane of phosphatidic acid.

    Science.gov (United States)

    Antonov, V F; Smirnova EYu; Shevchenko, E V

    1990-02-01

    The effect of the electric field on the phase transition temperature (Tc) of acidic 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA) and 1,2-dipalmitoyl-sn-glycero-3-thionphosphate (thion-DPPA) and zwitterion, i.e. 1,2-dipalmitoyl-rac-3-phosphocholine and 1,2-distearoyl-rac-glycero-3-phosphocholine (DPPC and DSPC), lipids has been investigated. The phase transition was detected using the jump-like increase effect in the conductance of the planar bilayer membrane. A voltage increase to 150 mV has been shown to increase the phase transition temperature in a bilayer lipid membrane (BLM) of phosphatidic acids (DPPA and thion-DPPA) by 8-12 degrees C while the transition temperature in the bilayer of zwitterion lipids (DPPC and DSPC) increases insignificantly. The increasing of Tt in BLM of acidic lipids is attributed to the voltage-induced changes in the molecule packing density. PMID:2340602

  3. Effect of Amphipathic HIV Fusion Inhibitor Peptides on POPC and POPC/Cholesterol Membrane Properties: A Molecular Simulation Study

    Directory of Open Access Journals (Sweden)

    Luís M. S. Loura

    2013-07-01

    Full Text Available T-20 and T-1249 fusion inhibitor peptides were shown to interact with 1-palmitoyl-2-oleyl-phosphatidylcholine (POPC (liquid disordered, ld and POPC/cholesterol (1:1 (POPC/Chol (liquid ordered, lo bilayers, and they do so to different extents. Although they both possess a tryptophan-rich domain (TRD, T-20 lacks a pocket binding domain (PBD, which is present in T-1249. It has been postulated that the PBD domain enhances FI interaction with HIV gp41 protein and with model membranes. Interaction of these fusion inhibitor peptides with both the cell membrane and the viral envelope membrane is important for function, i.e., inhibition of the fusion process. We address this problem with a molecular dynamics approach focusing on lipid properties, trying to ascertain the consequences and the differences in the interaction of T-20 and T-1249 with ld and lo model membranes. T-20 and T-1249 interactions with model membranes are shown to have measurable and different effects on bilayer structural and dynamical parameters. T-1249’s adsorption to the membrane surface has generally a stronger influence in the measured parameters. The presence of both binding domains in T-1249 appears to be paramount to its stronger interaction, and is shown to have a definite importance in membrane properties upon peptide adsorption.

  4. A Programmable DNA Origami Platform to Organize SNAREs for Membrane Fusion.

    Science.gov (United States)

    Xu, Weiming; Nathwani, Bhavik; Lin, Chenxiang; Wang, Jing; Karatekin, Erdem; Pincet, Frederic; Shih, William; Rothman, James E

    2016-04-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes are the core molecular machinery of membrane fusion, a fundamental process that drives inter- and intracellular communication and trafficking. One of the questions that remains controversial has been whether and how SNAREs cooperate. Here we show the use of self-assembled DNA-nanostructure rings to template uniform-sized small unilamellar vesicles containing predetermined maximal number of externally facing SNAREs to study the membrane-fusion process. We also incorporated lipid-conjugated complementary ssDNA as tethers into vesicle and target membranes, which enabled bypass of the rate-limiting docking step of fusion reactions and allowed direct observation of individual membrane-fusion events at SNARE densities as low as one pair per vesicle. With this platform, we confirmed at the single event level that, after docking of the templated-SUVs to supported lipid bilayers (SBL), one to two pairs of SNAREs are sufficient to drive fast lipid mixing. Modularity and programmability of this platform makes it readily amenable to studying more complicated systems where auxiliary proteins are involved. PMID:26938705

  5. Structure and membrane interaction of the internal fusion peptide of avian sarcoma leukosis virus.

    Science.gov (United States)

    Cheng, Shu-Fang; Wu, Cheng-Wei; Kantchev, Eric Assen B; Chang, Ding-Kwo

    2004-12-01

    The structure and membrane interaction of the internal fusion peptide (IFP) fragment of the avian sarcoma and leucosis virus (ASLV) envelope glycoprotein was studied by an array of biophysical methods. The peptide was found to induce lipid mixing of vesicles more strongly than the fusion peptide derived from the N-terminal fusion peptide of influenza virus (HA2-FP). It was observed that the helical structure was enhanced in association with the model membranes, particularly in the N-terminal portion of the peptide. According to the infrared study, the peptide inserted into the membrane in an oblique orientation, but less deeply than the influenza HA2-FP. Analysis of NMR data in sodium dodecyl sulfate micelle suspension revealed that Pro13 of the peptide was located near the micelle-water interface. A type II beta-turn was deduced from NMR data for the peptide in aqueous medium, demonstrating a conformational flexibility of the IFP in analogy to the N-terminal FP such as that of gp41. A loose and multimodal self-assembly was deduced from the rhodamine fluorescence self-quenching experiments for the peptide bound to the membrane bilayer. Oligomerization of the peptide and its variants can also be observed in the electrophoretic experiments, suggesting a property in common with other N-terminal FP of class I fusion proteins. PMID:15606759

  6. Fusion Pore Diameter Regulation by Cations Modulating Local Membrane Anisotropy

    Directory of Open Access Journals (Sweden)

    Doron Kabaso

    2012-01-01

    Full Text Available The fusion pore is an aqueous channel that is formed upon the fusion of the vesicle membrane with the plasma membrane. Once the pore is open, it may close again (transient fusion or widen completely (full fusion to permit vesicle cargo discharge. While repetitive transient fusion pore openings of the vesicle with the plasma membrane have been observed in the absence of stimulation, their frequency can be further increased using a cAMP-increasing agent that drives the opening of nonspecific cation channels. Our model hypothesis is that the openings and closings of the fusion pore are driven by changes in the local concentration of cations in the connected vesicle. The proposed mechanism of fusion pore dynamics is considered as follows: when the fusion pore is closed or is extremely narrow, the accumulation of cations in the vesicle (increased cation concentration likely leads to lipid demixing at the fusion pore. This process may affect local membrane anisotropy, which reduces the spontaneous curvature and thus leads to the opening of the fusion pore. Based on the theory of membrane elasticity, we used a continuum model to explain the rhythmic opening and closing of the fusion pore.

  7. Study of water diffusion on single-supported bilayer lipid membranes by quasielastic neutron scattering

    DEFF Research Database (Denmark)

    Bai, M.; Miskowiec, A.; Hansen, F. Y.;

    2012-01-01

    High-energy-resolution quasielastic neutron scattering has been used to elucidate the diffusion of water molecules in proximity to single bilayer lipid membranes supported on a silicon substrate. By varying sample temperature, level of hydration, and deuteration, we identify three different types...

  8. Effect of the dipole potential of a bilayer lipid membrane on gramicidin channel dissociation kinetics.

    OpenAIRE

    Rokitskaya, T I; Antonenko, Y N; Kotova, E A

    1997-01-01

    A technique of measuring of the light-induced transients of the gramicidin-mediated electric current across a membrane in the presence of a photosensitizer has been applied for the study of the effect of agents modifying the dipole potential of a bilayer lipid membrane (phloretin, 6-ketocholestanol, and RH421) on the processes of the gramicidin channel dissociation and formation. It is shown that phloretin, known to lower the dipole potential, decelerates the flash-induced decrease in the cur...

  9. Atomistic simulation of lipid and DiI dynamics in membrane bilayers under tension

    OpenAIRE

    Muddana, Hari S.; Gullapalli, Ramachandra R.; Manias, Evangelos; Butler, Peter J.

    2010-01-01

    Membrane tension modulates cellular processes by initiating changes in the dynamics of its molecular constituents. To quantify the precise relationship between tension, structural properties of the membrane, and the dynamics of lipids and a lipophilic reporter dye, we performed atomistic molecular dynamics (MD) simulations of DiI-labeled dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under physiological lateral tensions ranging from −2.6 mN m−1 to 15.9 mN m−1. Simulations showed that th...

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

    OpenAIRE

    Marianna Foldvari

    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 informa...

  11. Sendai Virus Fusion Activity as Modulated by Target Membrane Components

    OpenAIRE

    Nunes-Correia, Isabel; Ramalho-Santos, João; Maria C Pedroso de Lima

    1998-01-01

    We have studied the differences between erythrocytes and erythrocyte ghosts as target membranes for the study of Sendai virus fusion activity. Fusion was monitored continuously by fluorescence dequenching of R18-labeled virus. Experiments were carried out either with or without virus/target membrane prebinding. When Sendai virus was added directly to a erythrocyte/erythrocyte ghost suspension, fusion was always lower than that obtained when experiments were carried out with virus already boun...

  12. Membrane Fusion Induced by Small Molecules and Ions

    OpenAIRE

    Sutapa Mondal Roy; Munna Sarkar

    2011-01-01

    Membrane fusion is a key event in many biological processes. These processes are controlled by various fusogenic agents of which proteins and peptides from the principal group. The fusion process is characterized by three major steps, namely, inter membrane contact, lipid mixing forming the intermediate step, pore opening and finally mixing of inner contents of the cells/vesicles. These steps are governed by energy barriers, which need to be overcome to complete fusion. Structural reorganizat...

  13. Stochastic transport through carbon nanotubes in lipid bilayers and live cell membranes

    Science.gov (United States)

    Geng, Jia; Kim, Kyunghoon; Zhang, Jianfei; Escalada, Artur; Tunuguntla, Ramya; Comolli, Luis R.; Allen, Frances I.; Shnyrova, Anna V.; Cho, Kang Rae; Munoz, Dayannara; Wang, Y. Morris; Grigoropoulos, Costas P.; Ajo-Franklin, Caroline M.; Frolov, Vadim A.; Noy, Aleksandr

    2014-10-01

    There is much interest in developing synthetic analogues of biological membrane channels with high efficiency and exquisite selectivity for transporting ions and molecules. Bottom-up and top-down methods can produce nanopores of a size comparable to that of endogenous protein channels, but replicating their affinity and transport properties remains challenging. In principle, carbon nanotubes (CNTs) should be an ideal membrane channel platform: they exhibit excellent transport properties and their narrow hydrophobic inner pores mimic structural motifs typical of biological channels. Moreover, simulations predict that CNTs with a length comparable to the thickness of a lipid bilayer membrane can self-insert into the membrane. Functionalized CNTs have indeed been found to penetrate lipid membranes and cell walls, and short tubes have been forced into membranes to create sensors, yet membrane transport applications of short CNTs remain underexplored. Here we show that short CNTs spontaneously insert into lipid bilayers and live cell membranes to form channels that exhibit a unitary conductance of 70-100 picosiemens under physiological conditions. Despite their structural simplicity, these `CNT porins' transport water, protons, small ions and DNA, stochastically switch between metastable conductance substates, and display characteristic macromolecule-induced ionic current blockades. We also show that local channel and membrane charges can control the conductance and ion selectivity of the CNT porins, thereby establishing these nanopores as a promising biomimetic platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors.

  14. Structure and dynamics of water and lipid molecules in charged anionic DMPG lipid bilayer membranes

    Science.gov (United States)

    Rønnest, A. K.; Peters, G. H.; Hansen, F. Y.; Taub, H.; Miskowiec, A.

    2016-04-01

    Molecular dynamics simulations have been used to investigate the influence of the valency of counter-ions on the structure of freestanding bilayer membranes of the anionic 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) lipid at 310 K and 1 atm. At this temperature, the membrane is in the fluid phase with a monovalent counter-ion and in the gel phase with a divalent counter-ion. The diffusion constant of water as a function of its depth in the membrane has been determined from mean-square-displacement calculations. Also, calculated incoherent quasielastic neutron scattering functions have been compared to experimental results and used to determine an average diffusion constant for all water molecules in the system. On extrapolating the diffusion constants inferred experimentally to a temperature of 310 K, reasonable agreement with the simulations is obtained. However, the experiments do not have the sensitivity to confirm the diffusion of a small component of water bound to the lipids as found in the simulations. In addition, the orientation of the dipole moment of the water molecules has been determined as a function of their depth in the membrane. Previous indirect estimates of the electrostatic potential within phospholipid membranes imply an enormous electric field of 108-109 V m-1, which is likely to have great significance in controlling the conformation of translocating membrane proteins and in the transfer of ions and molecules across the membrane. We have calculated the membrane potential for DMPG bilayers and found ˜1 V (˜2 ṡ 108 V m-1) when in the fluid phase with a monovalent counter-ion and ˜1.4 V (˜2.8 ṡ 108 V m-1) when in the gel phase with a divalent counter-ion. The number of water molecules for a fully hydrated DMPG membrane has been estimated to be 9.7 molecules per lipid in the gel phase and 17.5 molecules in the fluid phase, considerably smaller than inferred experimentally for 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC

  15. Experimental evidence for hydrophobic matching and membrane-mediated interactions in lipid bilayers containing gramicidin.

    OpenAIRE

    Harroun, T A; Heller, W T; Weiss, T M; Yang, L; Huang, H W

    1999-01-01

    Hydrophobic matching, in which transmembrane proteins cause the surrounding lipid bilayer to adjust its hydrocarbon thickness to match the length of the hydrophobic surface of the protein, is a commonly accepted idea in membrane biophysics. To test this idea, gramicidin (gD) was embedded in 1, 2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and 1, 2-myristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers at the peptide/lipid molar ratio of 1:10. Circular dichroism (CD) was measured to ensure tha...

  16. Impact of monoolein on aquaporin1-based supported lipid bilayer membranes

    Science.gov (United States)

    Wang, Zhining; Wang, Xida; Ding, Wande; Wang, Miaoqi; Qi, Xin; Gao, Congjie

    2015-08-01

    Aquaporin (AQP) based biomimetic membranes have attracted considerable attention for their potential water purification applications. In this paper, AQP1 incorporated biomimetic membranes were prepared and characterized. The morphology and structure of the biomimetic membranes were characterized by in situ atomic force microscopy (AFM), infrared absorption spectroscopy, fluorescence microscopy, and contact angle measurements. The nanofiltration performance of the AQP1 incorporated membranes was investigated at 4 bar by using 2 g l-1 NaCl as feed solution. Lipid mobility plays an important role in the performance of the AQP1 incorporated supported lipid bilayer (SLB) membranes. We demonstrated that the lipid mobility is successfully tuned by the addition of monoolein (MO). Through in situ AFM and fluorescence recovery after photo-bleaching (FRAP) measurements, the membrane morphology and the molecular mobility were studied. The lipid mobility increased in the sequence DPPC rejection. This study may provide some useful insights for improving the water purification performance of biomimetic membranes.

  17. Folding of β-barrel membrane proteins in lipid bilayers - Unassisted and assisted folding and insertion.

    Science.gov (United States)

    Kleinschmidt, Jörg H

    2015-09-01

    In cells, β-barrel membrane proteins are transported in unfolded form to an outer membrane into which they fold and insert. Model systems have been established to investigate the mechanisms of insertion and folding of these versatile proteins into detergent micelles, lipid bilayers and even synthetic amphipathic polymers. In these experiments, insertion into lipid membranes is initiated from unfolded forms that do not display residual β-sheet secondary structure. These studies therefore have allowed the investigation of membrane protein folding and insertion in great detail. Folding of β-barrel membrane proteins into lipid bilayers has been monitored from unfolded forms by dilution of chaotropic denaturants that keep the protein unfolded as well as from unfolded forms present in complexes with molecular chaperones from cells. This review is aimed to provide an overview of the principles and mechanisms observed for the folding of β-barrel transmembrane proteins into lipid bilayers, the importance of lipid-protein interactions and the function of molecular chaperones and folding assistants. This article is part of a Special Issue entitled: Lipid-protein interactions. PMID:25983306

  18. Advances in the use of nanoscale bilayers to study membrane protein structure and function.

    Science.gov (United States)

    Malhotra, Ketan; Alder, Nathan N

    2014-10-01

    Within the last decade, nanoscale lipid bilayers have emerged as powerful experimental systems in the analysis of membrane proteins (MPs) for both basic and applied research. These discoidal lipid lamellae are stabilized by annuli of specially engineered amphipathic polypeptides (nanodiscs) or polymers (SMALPs/Lipodisqs®). As biomembrane mimetics, they are well suited for the reconstitution of MPs within a controlled lipid environment. Moreover, because they are water-soluble, they are amenable to solution-based biochemical and biophysical experimentation. Hence, due to their solubility, size, stability, and monodispersity, nanoscale lipid bilayers offer technical advantages over more traditional MP analytic approaches such as detergent solubilization and reconstitution into lipid vesicles. In this article, we review some of the most recent advances in the synthesis of polypeptide- and polymer-bound nanoscale lipid bilayers and their application in the study of MP structure and function. PMID:25023464

  19. Evidence for the “Cold Fusion” of Bilayer Membranes

    NARCIS (Netherlands)

    Ravoo, Bart Jan; Kevelam, Jan; Weringa, Wilke D.; Engberts, Jan B.F.N.

    1998-01-01

    Polymerization of the lipid headgroups inhibits calcium-induced fusion of small unilamellar vesicles of the lipid di-n-dodecyloxypropyl beta-nitrostyryl phosphate but does not influence vesicle aggregation. Addition of a copolymer of lauryl methacrylate and acrylamide (LMPAM) provides the vesicles w

  20. pH regulation of amphotericin B channels activity in the bilayer lipid membrane

    Science.gov (United States)

    Shahmoradi, Tahereh; Sepehry, Hamid; Ashrafpour, Manuchehr

    2016-01-01

    Background: Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of systemic fungal infections in spite of its secondary effects. The pH plays a crucial role in modulating biophysical features of ion channels in the bilayer lipid membranes. Aim: In this study, the role of pH in the regulation of AmB channel was assessed by single channel recording of ion channel incorporated in the artificial membrane. Materials and Methods: Bilayer lipid membrane was formed by phosphatidylcholine in a 350 μm diameter aperture between two chambers, cis and trans contained 200/50 mMKCl solutions, respectively; then AmB was incorporated into the bilayer lipid membrane. Single channel recordings were used to indicate the effects of pH changes on AmB channels activity. The records were analyzed by Clamp fit 10 software. Results: A kinetic analysis of single channel currents indicated a cation ion channel with 500 pS conductance and voltage-dependence of the open probability of the AmB channel (Po). A reduction of cis pH to 6 decreased Po and conductance. This effect was also voltage-dependent, being greater at a more positive above −40. The pH changes in the range of 6-8 had no effect on the reversal potential and ion selectivity. Conclusion: Our data indicated that extracellular acidity can reduce AmB activity. PMID:27003977

  1. Modeling Lipid-Lipid Correlations across a Bilayer Membrane Using the Quasi-chemical Approximation.

    Science.gov (United States)

    Bossa, Guilherme Volpe; Roth, Joseph; May, Sylvio

    2015-09-15

    Mixed fluid-like lipid membranes exhibit interactions not only among the lipids within a given leaflet but also across the bilayer. The ensuing collective interleaflet coupling of entire membrane domains has been modeled previously using various mean-field approaches. Yet, also on the level of individual lipids have correlations across the bilayer been observed experimentally for binary mixtures of charged/uncharged lipids with mismatching combinations of short and long acyl chain lengths. The present study proposes a lattice gas model to quantify these correlations. To this end, we represent a macroscopically homogeneous lipid bilayer by two coupled two-dimensional lattice gases that we study using the quasi-chemical approximation. We demonstrate that the rationalization of previous experimental results is only possible if besides two-body lipid-lipid interactions within and across the bilayer our model also accounts for an additional multibody interaction mechanism, namely the local hydrophobic height mismatch created by pairing short and long chain lipids together. The robustness of the quasi-chemical approximation is verified by comparison with Monte Carlo simulations. PMID:26302019

  2. Affinity of four polar neurotransmitters for lipid bilayer membranes

    DEFF Research Database (Denmark)

    Wang, Chunhua; Ye, Fengbin; Valardez, Gustavo F.;

    2011-01-01

    Weak interactions of neurotransmitters and the lipid matrix in the synaptic membrane have been hypothesized to play a role in synaptic transmission of nerve signals, particularly with respect to receptor desensitization (Cantor, R. S. Biochemistry 2003, 42, 11891). The strength of such interactions......, however, was not measured, and this is an obvious impediment for further evaluation and understanding of a possible role for desensitization. We have used dialysis equilibrium to directly measure the net affinity of selected neurotransmitters for lipid membranes and analyzed this affinity data with...... respect to calorimetric measurements and molecular dynamics simulations. We studied an anionic (glutamate), a cationic (acetylcholine), and two zwitterionic (-aminobutyric acid and glycine) neurotransmitters, and membranes of pure dimyristoyl phosphatidylcholine (DMPC), DMPC doped with 10% anionic lipid...

  3. Lipid Bilayer Domain Fluctuations as a Probe of Membrane Viscosity

    Science.gov (United States)

    Camley, Brian A.; Esposito, Cinzia; Baumgart, Tobias; Brown, Frank L.H.

    2010-01-01

    We argue that membrane viscosity, ηm, plays a prominent role in the thermal fluctuation dynamics of micron-scale lipid domains. A theoretical expression is presented for the timescales of domain shape relaxation, which reduces to the well-known ηm = 0 result of Stone and McConnell in the limit of large domain sizes. Experimental measurements of domain dynamics on the surface of ternary phospholipid and cholesterol vesicles confirm the theoretical results and suggest domain flicker spectroscopy as a convenient means to simultaneously measure both the line tension, σ, and the membrane viscosity, ηm, governing the behavior of individual lipid domains. PMID:20858410

  4. Molecular dynamics simulations of the interactions of medicinal plant extracts and drugs with lipid bilayer membranes

    DEFF Research Database (Denmark)

    Kopec, Wojciech; Telenius, Jelena; Khandelia, Himanshu

    2013-01-01

    Several small drugs and medicinal plant extracts, such as the Indian spice extract curcumin, have a wide range of useful pharmacological properties that cannot be ascribed to binding to a single protein target alone. The lipid bilayer membrane is thought to mediate the effects of many such...... molecules directly via perturbation of the plasma membrane structure and dynamics, or indirectly by modulating transmembrane protein conformational equilibria. Furthermore, for bioavailability, drugs must interact with and eventually permeate the lipid bilayer barrier on the surface of cells. Biophysical...... studies of the interactions of drugs and plant extracts are therefore of interest. Molecular dynamics simulations, which can access time and length scales that are not simultaneously accessible by other experimental methods, are often used to obtain quantitative molecular and thermodynamic descriptions of...

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

    International Nuclear Information System (INIS)

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

  6. Nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor

    DEFF Research Database (Denmark)

    Borch, Jonas; Torta, Federico; Sligar, Stephen G; Roepstorff, Peter

    2008-01-01

    Nanodiscs are self-assembled soluble discoidal phospholipids bilayers encirculated by an amphipathic protein that together provide a functional stabilized membrane disk for the incorporation of membrane-bound and membrane-associated molecules. The scope of the present work is to investigate how n...

  7. Measurement of voltage dependence of capacitance of planar bilayer lipid membrane with a patch clamp amplifier.

    OpenAIRE

    Toyama, S.; Nakamura, A; Toda, F

    1991-01-01

    The voltage dependence of capacitance was measured by using the setup which was almost the same as that for the study of ion channels. The coefficient which represents the voltage dependence of capacitance itself also changes as a function of the duration of voltage application if hexadecane is contained in bilayer lipid membrane (BLM). The method of Alvarez, O., and R. Latorre (1978. Biophys. J. 21:1-17) was extended to treat BLM with hexadecane.

  8. Atomistic simulation of lipid and DiI dynamics in membrane bilayers under tension.

    Science.gov (United States)

    Muddana, Hari S; Gullapalli, Ramachandra R; Manias, Evangelos; Butler, Peter J

    2011-01-28

    Membrane tension modulates cellular processes by initiating changes in the dynamics of its molecular constituents. To quantify the precise relationship between tension, structural properties of the membrane, and the dynamics of lipids and a lipophilic reporter dye, we performed atomistic molecular dynamics (MD) simulations of DiI-labeled dipalmitoylphosphatidylcholine (DPPC) lipid bilayers under physiological lateral tensions ranging from -2.6 mN m(-1) to 15.9 mN m(-1). Simulations showed that the bilayer thickness decreased linearly with tension consistent with volume-incompressibility, and this thinning was facilitated by a significant increase in acyl chain interdigitation at the bilayer midplane and spreading of the acyl chains. Tension caused a significant drop in the bilayer's peak electrostatic potential, which correlated with the strong reordering of water and lipid dipoles. For the low tension regime, the DPPC lateral diffusion coefficient increased with increasing tension in accordance with free-area theory. For larger tensions, free area theory broke down due to tension-induced changes in molecular shape and friction. Simulated DiI rotational and lateral diffusion coefficients were lower than those of DPPC but increased with tension in a manner similar to DPPC. Direct correlation of membrane order and viscosity near the DiI chromophore, which was just under the DPPC headgroup, indicated that measured DiI fluorescence lifetime, which is reported to decrease with decreasing lipid order, is likely to be a good reporter of tension-induced decreases in lipid headgroup viscosity. Together, these results offer new molecular-level insights into membrane tension-related mechanotransduction and into the utility of DiI in characterizing tension-induced changes in lipid packing. PMID:21152516

  9. Defining the Free-Energy Landscape of Curvature-Inducing Proteins on Membrane Bilayers

    CERN Document Server

    Tourdot, Richard W; Radhakrishnan, Ravi

    2015-01-01

    Curvature-sensing and curvature-remodeling proteins are known to reshape cell membranes, and this remodeling event is essential for key biophysical processes such as tubulation, exocytosis, and endocytosis. Curvature-inducing proteins can act as curvature sensors as well as induce curvature in cell membranes to stabilize emergent high curvature, non-spherical, structures such as tubules, discs, and caveolae. A definitive understanding of the interplay between protein recruitment and migration, the evolution of membrane curvature, and membrane morphological transitions is emerging but remains incomplete. Here, within a continuum framework and using the machinery of Monte Carlo simulations, we introduce and compare three free-energy methods to delineate the free-energy landscape of curvature-inducing proteins on bilayer membranes. We demonstrate the utility of the Widom test-particle/field insertion methodology in computing the excess chemical potentials associated with curvature-inducing proteins on the membra...

  10. A New Method for Measuring Edge Tensions and Stability of Lipid Bilayers: Effect of Membrane Composition

    CERN Document Server

    Portet, Thomas; 10.1016/j.bpj.2010.09.032

    2011-01-01

    We report a new and facile method for measuring edge tensions of lipid membranes. The approach is based on electroporation of giant unilamellar vesicles and analysis of the pore closure dynamics. We applied this method to evaluate the edge tension in membranes with four different compositions: egg phosphatidylcholine (EggPC), dioleoylphosphatidylcholine (DOPC), and mixtures of the latter with cholesterol and dioleoylphosphatidylethanolamine (DOPE). Our data confirm previous results for EggPC and DOPC. The addition of 17 mol % cholesterol to the DOPC membrane causes an increase in the membrane edge tension. On the contrary, when the same fraction of DOPE is added to the membrane, a decrease in the edge tension is observed, which is an unexpected result considering the inverted-cone shape geometry of the molecule. Presumably, interlipid hydrogen bonding lies in the origin of this behavior. Furthermore, cholesterol was found to lower the lysis tension of DOPC bilayers. This behavior differs from that observed on...

  11. Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

    Energy Technology Data Exchange (ETDEWEB)

    Eddy, Matthew T. [The Scripps Research Institute, Department of Integrative Structural and Computational Biology (United States); Su, Yongchao; Silvers, Robert; Andreas, Loren; Clark, Lindsay [Massachusetts Institute of Technology, Department of Chemistry (United States); Wagner, Gerhard [Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology (United States); Pintacuda, Guido; Emsley, Lyndon [Université de Lyon, Centre de RMN à Très Hauts Champs, Institut des Sciences Analytiques (CNRS, ENS Lyon, UCB Lyon 1) (France); Griffin, Robert G., E-mail: rgg@mit.edu [Massachusetts Institute of Technology, Department of Chemistry (United States)

    2015-04-15

    The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for {sup 13}C line widths and <0.5 ppm {sup 15}N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the

  12. Lipid bilayer-bound conformation of an integral membrane beta barrel protein by multidimensional MAS NMR

    International Nuclear Information System (INIS)

    The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5–0.3 ppm for 13C line widths and <0.5 ppm 15N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported

  13. Properties of ganglioside GM1 in phosphatidylcholine bilayer membranes.

    OpenAIRE

    Reed, R A; Shipley, G G

    1996-01-01

    Gangliosides have been shown to function as cell surface receptors, as well as participating in cell growth, differentiation, and transformation. In spite of their multiple biological functions, relatively little is known about their structure and physical properties in membrane systems. The thermotropic and structural properties of ganglioside GM1 alone and in a binary system with 1,2-dipalmitoyl phosphatidylcholine (DPPC) have been investigated by differential scanning calorimetry (DSC) and...

  14. Ion-channel-like behavior in lipid bilayer membranes at the melting transition

    CERN Document Server

    Gallaher, Jill; Heimburg, Thomas; Bier, Martin

    2010-01-01

    It is well known that at the gel-liquid phase transition temperature a lipid bilayer membrane exhibits an increased ion permeability. We analyze the quantized currents in which the increased permeability presents itself. The open time histogram shows a "-3/2" power law which implies an open-closed transition rate that decreases like $k(t) \\propto t^{-1}$ as time evolves. We propose a "pore freezing" model to explain the observations. We discuss how this model also leads to the $1/f^{\\alpha}$ noise that is commonly observed in currents across biological and artificial membranes.

  15. Analysis of Ion Transport through a Single Channel of Gramicidin A in Bilayer Lipid Membranes.

    Science.gov (United States)

    Kubota, Shintaro; Shirai, Osamu; Kitazumi, Yuki; Kano, Kenji

    2016-01-01

    Ion transport through a single channel of gramicidin A (GA) within the bilayer lipid membrane (BLM) between two aqueous phases (W1 and W2) has been analyzed based on the electroneutrality principle. The single-channel current increases in proportion to the magnitude of the applied membrane potential and is also dependent on the permeability coefficients of electrolyte ions (K(+) and Cl(-)). By varying the ratio of the concentration of KCl in W1 to that in W2, the ratio of the diffusion coefficient of K(+) in the BLM to that of Cl(-) in the BLM can be evaluated. PMID:26860564

  16. Salt-bridge-supported bilayer lipid membrane biosensor for determination of anticancer drug cyclophosphamide

    Science.gov (United States)

    Zhang, Yanli; Wang, Tao; Zhang, Chunxu; Shen, Hanxi; Chao, Fuhuan

    2001-09-01

    A novel biosensor for assaying anticancer drug cyclophosphamide was constructed with salt-bridge supported bilayer lipid membrane modified with tetraphenylborate- cyclophosphamide complex. The modification was achieved by the introduction of the complex into the membrane forming solution. The biosensor show a linear response to the drug over the concentration range 8.96 X 10-6 mol L-1. The effects of coexistent substances and pH on assay were evaluated. The results show that the distinguish merits of this kind of biosensor is the excellently biological compatibility and no need of mediator for ions exchange. It also shows good selectivity and sensitivity for cyclophosphamide assay.

  17. Bathroom greywater recycling using polyelectrolyte-complex bilayer membrane: Advanced study of membrane structure and treatment efficiency.

    Science.gov (United States)

    Oh, K S; Poh, P E; Chong, M N; Chan, E S; Lau, E V; Saint, C P

    2016-09-01

    Polyelectrolyte-complex bilayer membrane (PCBM) was fabricated using biodegradable chitosan and alginate polymers for subsequent application in the treatment of bathroom greywater. In this study, the properties of PCBMs were studied and it was found that the formation of polyelectrolyte network reduced the molecular weight cut-off (MWCO) from 242kDa in chitosan membrane to 2.71kDa in PCBM. The decrease in MWCO of PCBM results in better greywater treatment efficiency, subsequently demonstrated in a greywater filtration study where treated greywater effluent met the household reclaimed water standard of <2 NTU turbidity and <30ppm total suspended solids (TSS). In addition, a further 20% improvement in chemical oxygen demand (COD) removal was achieved as compared to a single layer chitosan membrane. Results from this study show that the biodegradable PCBM is a potential membrane material in producing clean treated greywater for non-potable applications. PMID:27185127

  18. Defining the free-energy landscape of curvature-inducing proteins on membrane bilayers

    Science.gov (United States)

    Tourdot, Richard W.; Ramakrishnan, N.; Radhakrishnan, Ravi

    2014-08-01

    Curvature-sensing and curvature-remodeling proteins, such as Amphiphysin, Epsin, and Exo70, are known to reshape cell membranes, and this remodeling event is essential for key biophysical processes such as tubulation, exocytosis, and endocytosis. Curvature-inducing proteins can act as curvature sensors; they aggregate to membrane regions matching their intrinsic curvature; as well as induce curvature in cell membranes to stabilize emergent high curvature, nonspherical, structures such as tubules, discs, and caveolae. A definitive understanding of the interplay between protein recruitment and migration, the evolution of membrane curvature, and membrane morphological transitions is emerging but remains incomplete. Here, within a continuum framework and using the machinery of Monte Carlo simulations, we introduce and compare three free-energy methods to delineate the free-energy landscape of curvature-inducing proteins on bilayer membranes. We demonstrate the utility of the Widom test particle (or field) insertion methodology in computing the excess chemical potentials associated with curvature-inducing proteins on the membrane—in particular, we use this method to track the onset of morphological transitions in the membrane at elevated protein densities. We validate this approach by comparing the results from the Widom method with those of thermodynamic integration and Bennett acceptance ratio methods. Furthermore, the predictions from the Widom method have been tested against analytical calculations of the excess chemical potential at infinite dilution. Our results are useful in precisely quantifying the free-energy landscape, and also in determining the phase boundaries associated with curvature-induction, curvature-sensing, and morphological transitions. This approach can be extended to studies exploring the role of thermal fluctuations and other external (control) variables, such as membrane excess area, in shaping curvature-mediated interactions on bilayer

  19. Maleimide-functionalized lipids that anchor polypeptides to lipid bilayers and membranes.

    Science.gov (United States)

    Elliott, J T; Prestwich, G D

    2000-01-01

    Two maleimide-containing diacylglycerol derivatives were synthesized to permit the anchoring of short peptides and longer polypeptides to phospholipid bilayers and membranes. The maleimide was introduced at the site normally occupied by a phospholipid headgroup. The first lipid, the dipalmitoyl ester of 1-maleimido-2,3-propanediol, was developed as a membrane anchor for extracellular domains of transmembrane proteins. The second anchoring lipid, in which the 3-position contained a 6-aminohexanoate, was designed for convenient modification with amine-reactive reporter groups. Specifically, the NBD fluorophore, 7-nitrobenzo-2-oxa-1, 3-diazole-aminohexanoic-N-hydroxysuccinimide ester, was attached to give an fluorescent anchoring reagent. Next, these reagents were applied to the anchoring of a C-terminally cysteamine-modified 8 kDa polypeptide that comprises the extracellular N-terminal domain of the human thrombin receptor, a transmembrane protease-activated receptor (PAR-1). Gel filtration and fluorescence analysis showed that the fluorescent lipopolypeptide spontaneously inserted into preformed phospholipid vesicles, but it did not insert into whole cell membranes. In contrast, the dipalmitoyl derivative could only be reconstituted into artificial membranes by mixing the lipopolypeptide and phospholipid before vesicle formation. These results suggest that biophysical interactions governing the lipopolypeptide insertion into artificial and cellular membranes may differ. The thiol-reactive lipidating reagents should be valuable materials for studying the structure and function of peptides and polypeptides at phospholipid bilayer surfaces. PMID:11087332

  20. Highly efficient integration of the viral portal proteins from different types of phages into planar bilayers for the black lipid membrane analysis.

    Science.gov (United States)

    Jing, Peng; Paraiso, Hallel; Burris, Benjamin

    2016-02-01

    The planar lipid bilayer technology is a technique that yields incredibly useful structural function information about a single channel protein. It is also currently actively utilized as a powerful platform using biological protein nanopores for the development of single-molecule nanopore sensing technology, as well as ultrafast DNA sequencing technology. The portal protein, GP10, from the bacteriophage Φ29 was the first phage portal protein shown to be successfully inserted into planar bilayer membranes, thereby it may inspire more researchers to apply the techniques to portal proteins from the other bacteriophages. However, the technology is far from perfect since the insertion of the channel proteins into planar bilayer membranes is not only technically difficult but also time-consuming. For the fusion of phage portal proteins, vesicles are typically needed to be reconstituted with the portal proteins to form proteoliposomes. However, most of the phage portal proteins have low solubility, and may self-aggregate during the preparation of the proteoliposomes. Furthermore, the fusion of the formed proteoliposomes is sporadic, unpredictable and varied from person to person. Due to the lack of experimental consistency between labs, the results from different methodologies reported for generating fusible proteoliposomes are highly variable. In this research, we propose a new method for the preparation of the fusible proteoliposomes containing portal proteins from bacteriophages, to circumvent the problems aforementioned. Compared to the conventional methods, this method was able to avoid the protein aggregation issues during the vesicle preparation by eliminating the need for detergents and the subsequent time-consuming step for detergent removal. The proteoliposomes prepared by the method were shown to be more efficiently and rapidly inserted into planar bilayer membranes bathed in different conducting buffer solutions including those with nonelectrolytes such as

  1. Membrane-Protein Interactions in a Generic Coarse-Grained Model for Lipid Bilayers

    CERN Document Server

    West, Beate; Schmid, Friederike

    2008-01-01

    We study membrane-protein interactions and membrane-mediated protein-protein interactions by Monte Carlo simulations of a generic coarse-grained model for lipid bilayers with cylindrical hydrophobic inclusions. The strength of the hydrophobic force and the hydrophobic thickness of the proteins are systematically varied. The results are compared with analytical predictions of two popular analytical theories: The Landau-de Gennes theory and the elastic theory. The elastic theory provides an excellent description of the fluctuation spectra of pure membranes and successfully reproduces the deformation profiles of membranes around single proteins. However, its prediction for the potential of mean force between proteins is not compatible with the simulation data for large distances. The simulations show that the lipid-mediated interactions are governed by five competing factors: Direct interactions, lipid-induced depletion interactions, lipid bridging, lipid packing, and a smooth long-range contribution. The mechan...

  2. Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

    Directory of Open Access Journals (Sweden)

    Daniel L Glauser

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

  3. Distribution and dynamics of quinones in the lipid bilayer mimicking the inner membrane of mitochondria.

    Science.gov (United States)

    Kaurola, Petri; Sharma, Vivek; Vonk, Amanda; Vattulainen, Ilpo; Róg, Tomasz

    2016-09-01

    Quinone and its analogues (Q) constitute an important class of compounds that perform key electron transfer reactions in oxidative- and photo-phosphorylation. In the inner membrane of mitochondria, ubiquinone molecules undergo continuous redox transitions enabling electron transfer between the respiratory complexes. In such a dynamic system undergoing continuous turnover for ATP synthesis, an uninterrupted supply of substrate molecules is absolutely necessary. In the current work, we have performed atomistic molecular dynamics simulations and free energy calculations to assess the structure, dynamics, and localization of quinone and its analogues in a lipid bilayer, whose composition mimics the one in the inner mitochondrial membrane. The results show that there is a strong tendency of both quinone and quinol molecules to localize in the vicinity of the lipids' acyl groups, right under the lipid head group region. Additionally, we observe a second location in the middle of the bilayer where quinone molecules tend to stabilize. Translocation of quinone through a lipid bilayer is very fast and occurs in 10-100ns time scale, whereas the translocation of quinol is at least an order of magnitude slower. We suggest that this has important mechanistic implications given that the localization of Q ensures maximal occupancy of the Q-binding sites or Q-entry points in electron transport chain complexes, thereby maintaining an optimal turnover rate for ATP synthesis. PMID:27342376

  4. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes.

    Science.gov (United States)

    Ryu, Yong-Sang; Wittenberg, Nathan J; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N; Lee, Sin-Doo

    2016-01-01

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

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

    International Nuclear Information System (INIS)

    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.

  6. Bilayer registry in a multicomponent asymmetric membrane : dependence on lipid composition and chain length

    CERN Document Server

    Polley, Anirban; Rao, Madan

    2013-01-01

    A question of considerable interest to cell membrane biology is whether phase segregated domains across an asymmetric bilayer are strongly correlated with each other and whether phase segregation in one leaflet can induce segregation in the other. We answer both these questions in the affirmative, using an atomistic molecular dynamics simulation to study the equilibrium statistical properties of a 3-component {\\em asymmetric} lipid bilayer comprising an unsaturated POPC (palmitoyl-oleoyl-phosphatidyl-choline), a saturated SM (sphingomyelin) and cholesterol with different composition ratios. Our simulations are done by fixing the composition of the upper leaflet to be at the coexistence of the liquid ordered ($l_o$) - liquid disordered ($l_d$) phases, while the composition of the lower leaflet is varied from the phase coexistence regime to the mixed $l_d$ phase, across a first-order phase boundary. In the regime of phase coexistence in each leaflet, we find strong transbilayer correlations of the $l_o$ domains...

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

    Science.gov (United States)

    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.

  8. Molecular View of the Role of Fusion Peptides in Promoting Positive Membrane Curvature

    NARCIS (Netherlands)

    Fuhrmans, Marc; Marrink, Siewert J.

    2012-01-01

    Fusion peptides are moderately hydrophobic segments of viral and nonviral membrane fusion proteins that enable these proteins to fuse two closely apposed biological membranes. In vitro assays furthermore show that even isolated fusion peptides alone can support membrane fusion in model systems. In a

  9. Thermodynamic Free Energy Methods to Investigate Shape Transitions In Bilayer Membranes

    CERN Document Server

    Ramakrishnan, N; Radhakrishnan, Ravi

    2015-01-01

    The conformational free energy landscape of a system is a fundamental thermodynamic quantity of importance particularly in the study of soft matter and biological systems, in which the entropic contributions play a dominant role. While computational methods to delineate the free energy landscape are routinely used to analyze the relative stability of conformational states, to determine phase boundaries, and to compute ligand-receptor binding energies its use in problems involving the cell membrane is limited. Here, we present an overview of four different free energy methods to study morphological transitions in bilayer membranes, induced either by the action of curvature remodeling proteins or due to the application of external forces. Using a triangulated surface as a model for the cell membrane and using the framework of dynamical triangulation Monte Carlo, we have focused on the methods of Widom insertion, thermodynamic integration, Bennett acceptance scheme, and umbrella sampling and weighted histogram a...

  10. Molecular View of the Role of Fusion Peptides in Promoting Positive Membrane Curvature

    OpenAIRE

    Fuhrmans, Marc; Marrink, Siewert J.

    2012-01-01

    Fusion peptides are moderately hydrophobic segments of viral and nonviral membrane fusion proteins that enable these proteins to fuse two closely apposed biological membranes. In vitro assays furthermore show that even isolated fusion peptides alone can support membrane fusion in model systems. In addition, the fusion peptides have a distinct effect on the phase diagram of lipid mixtures. Here, we present molecular dynamics simulations investigating the effect of a particular fusion peptide, ...

  11. Investigation of SNARE-Mediated Membrane Fusion Mechanism Using Atomic Force Microscopy

    OpenAIRE

    Abdulreda, Midhat H.; Moy, Vincent T.

    2009-01-01

    Membrane fusion is driven by specialized proteins that reduce the free energy penalty for the fusion process. In neurons and secretory cells, soluble N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptors (SNAREs) mediate vesicle fusion with the plasma membrane during vesicular content release. Although, SNAREs have been widely accepted as the minimal machinery for membrane fusion, the specific mechanism for SNARE-mediated membrane fusion remains an active area of research. Her...

  12. Molecular dynamics study of lipid bilayers modeling the plasma membranes of mouse hepatocytes and hepatomas

    Science.gov (United States)

    Andoh, Yoshimichi; Aoki, Noriyuki; Okazaki, Susumu

    2016-02-01

    Molecular dynamics (MD) calculations of lipid bilayers modeling the plasma membranes of normal mouse hepatocytes and hepatomas in water have been performed under physiological isothermal-isobaric conditions (310.15 K and 1 atm). The changes in the membrane properties induced by hepatic canceration were investigated and were compared with previous MD calculations included in our previous study of the changes in membrane properties induced by murine thymic canceration. The calculated model membranes for normal hepatocytes and hepatomas comprised 23 and 24 kinds of lipids, respectively. These included phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. We referred to previously published experimental values for the mole fraction of the lipids adopted in the present calculations. The calculated structural and dynamic properties of the membranes such as lateral structure, order parameters, lateral self-diffusion constants, and rotational correlation times all showed that hepatic canceration causes plasma membranes to become more ordered laterally and less fluid. Interestingly, this finding contrasts with the less ordered structure and increased fluidity of plasma membranes induced by thymic canceration observed in our previous MD study.

  13. Interaction of pyridinium bis-retinoid (A2E) with bilayer lipid membranes.

    Science.gov (United States)

    Sokolov, V S; Sokolenko, E A; Sokolov, A V; Dontsov, A E; Chizmadzhev, Y A; Ostrovsky, M A

    2007-02-01

    The accumulation of lipofuscin granules within the retinal pigment epithelium (RPE) cells is correlated with the progression of age-related macular degeneration. One of the fluorophores contained in lipofiscin granules is pyridinium bis-retinoid (A2E). To test its membrane-toxic effect, the interaction of A2E with bilayer lipid membranes (BLM) was studied. The incorporation of charged A2E molecules into the membranes has been detected as a change of either zeta-potential of multilayer liposomes or boundary potential of BLM. It was shown that the presence of up to 25mol% of A2E did not destabilize the bilayers made of saturated phosphatidylcholine (PC). However, the destabilizing effect became very significant when BLM contained negatively charged lipids such as cardiolipin or phosphatidylserine. The electrical breakdown measurements revealed that the A2E-induced decrease of BLM stability was primarily associated with the growing probability of lipid pore formation. It was found from the measurements of boundary potential of BLM that exposure of A2E to light initiates its transformation into at least two products. One of them is epoxy-A2E, which, being hydrophilic, moves from the membrane into water solution. The other product is a non-identified hydrophobic substance. Illumination of A2E-containing BLM made from unsaturated PC by visible light caused the membrane damage presumably due to oxidation of these lipids by singlet oxygen generated by excited A2E molecules. However, this effect was very weak compared to the effect of known photosensitizers. The illumination of BLM with A2E also leads to the damage of gramicidin incorporated into the membrane, as was detected by measuring the conductance of channels formed by this peptide. PMID:17070694

  14. Inter-layer slide and stress relaxation in a bilayer fluid membrane in the patch-clamp setting

    OpenAIRE

    Mukhin, Sergei I.; Baoukina, Svetlana V.

    2003-01-01

    Protein mechanosensitive channels (MS) are activated by tension transmitted through the lipid bilayer. We propose a theory of lateral stress relaxation in a bilayer lipid membrane exposed to external pressure pulse in the patch-clamp experimental setting. It is shown that transfer of lipid molecules into a strained region is thermodynamically advantageous due to local decrease of the stress. Considered stress relaxation mechanism may explain recent experimental observations (Davidson and Mart...

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

    Science.gov (United States)

    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.

  16. Determining the orientation of protegrin-1 in DLPC bilayers using an implicit solvent-membrane model.

    Directory of Open Access Journals (Sweden)

    Abdallah Sayyed-Ahmad

    Full Text Available Continuum models that describe the effects of solvent and biological membrane molecules on the structure and behavior of antimicrobial peptides, holds a promise to improve our understanding of the mechanisms of antimicrobial action of these peptides. In such methods, a lipid bilayer model membrane is implicitly represented by multiple layers of relatively low dielectric constant embedded in a high dielectric aqueous solvent, while an antimicrobial peptide is accounted for by a dielectric cavity with fixed partial charge at the center of each one of its atoms. In the present work, we investigate the ability of continuum approaches to predict the most probable orientation of the beta-hairpin antimicrobial peptide Protegrin-1 (PG-1 in DLPC lipid bilayers by calculating the difference in the transfer free energy from an aqueous environment to a membrane-water environment for multiple orientations. The transfer free energy is computed as a sum of two terms; polar/electrostatic and non-polar. They both include energetic and entropic contributions to the free energy. We numerically solve the Poisson-Boltzmann equation to calculate the electrostatic contribution to the transfer free energy, while the non-polar contribution to the free energy is approximated using a linear solvent accessible surface area relationships. The most probable orientation of PG-1 is that with the lowest relative transfer free energy. Our simulation results indicate that PG-1 assumes an oblique orientation in DLPC lipid bilayers. The predicted most favorable orientation was with a tilt angle of 19 degrees, which is in qualitative agreement with the experimentally observed orientations derived from solid-state NMR data.

  17. Bilayer porous scaffold based on poly-(ɛ-caprolactone) nanofibrous membrane and gelatin sponge for favoring cell proliferation

    Science.gov (United States)

    Zhou, Zhihua; Zhou, Yang; Chen, Yiwang; Nie, Huarong; Wang, Yang; Li, Fan; Zheng, Yan

    2011-12-01

    Electrospun poly-(ɛ-caprolactone) (PCL) nanofibers has been widely used in the medical prosthesis. However, poor hydrophilicity and the lack of natural recognition sites for covalent cell-recognition signal molecules to promote cell attachment have limited its utility as tissue scaffolds. In this study, Bilayer porous scaffolds based on PCL electrospun membranes and gelatin (GE) sponges were fabricated through soft hydrolysis of PCL electrospun followed by grafting gelatin onto the fiber surface, through crosslinking and freeze drying treatment of additional gelatin coat and grafted gelatin surface. GE sponges were stably anchored on PCL membrane surface with the aid of grafted GE molecules. The morphologies of bilayer porous scaffolds were observed through SEM. The contact angle of the scaffolds was 0°, the mechanical properties of scaffolds were measured by tensile test, Young's moduli of PCL scaffolds before and after hydrolysis are 66-77.3 MPa and 62.3-75.4 MPa, respectively. Thus, the bilayer porous scaffolds showed excellent hydrophilic surface and desirable mechanical strength due to the soft hydrolysis and GE coat. The cell culture results showed that the adipose derived mesenchymal stem cells did more favor to adhere and grow on the bilayer porous scaffolds than on PCL electrospun membranes. The better cell affinity of the final bilayer scaffolds not only attributed to the surface chemistry but also the introduction of bilayer porous structure.

  18. Bilayer porous scaffold based on poly-(ε-caprolactone) nanofibrous membrane and gelatin sponge for favoring cell proliferation

    International Nuclear Information System (INIS)

    Electrospun poly-(ε-caprolactone) (PCL) nanofibers has been widely used in the medical prosthesis. However, poor hydrophilicity and the lack of natural recognition sites for covalent cell-recognition signal molecules to promote cell attachment have limited its utility as tissue scaffolds. In this study, Bilayer porous scaffolds based on PCL electrospun membranes and gelatin (GE) sponges were fabricated through soft hydrolysis of PCL electrospun followed by grafting gelatin onto the fiber surface, through crosslinking and freeze drying treatment of additional gelatin coat and grafted gelatin surface. GE sponges were stably anchored on PCL membrane surface with the aid of grafted GE molecules. The morphologies of bilayer porous scaffolds were observed through SEM. The contact angle of the scaffolds was 0°, the mechanical properties of scaffolds were measured by tensile test, Young's moduli of PCL scaffolds before and after hydrolysis are 66-77.3 MPa and 62.3-75.4 MPa, respectively. Thus, the bilayer porous scaffolds showed excellent hydrophilic surface and desirable mechanical strength due to the soft hydrolysis and GE coat. The cell culture results showed that the adipose derived mesenchymal stem cells did more favor to adhere and grow on the bilayer porous scaffolds than on PCL electrospun membranes. The better cell affinity of the final bilayer scaffolds not only attributed to the surface chemistry but also the introduction of bilayer porous structure.

  19. Bilayer porous scaffold based on poly-({epsilon}-caprolactone) nanofibrous membrane and gelatin sponge for favoring cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Zhou Zhihua; Zhou Yang [Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Chen Yiwang, E-mail: ywchen@ncu.edu.cn [Department of Chemistry, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Nie Huarong, E-mail: niehr@iccas.ac.cn [Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China); Wang Yang [First Affiliated Hospital, Nanchang University, 17 Yongwaizheng Road, Nanchang 330006 (China); Li Fan; Zheng Yan [Institute of Polymers, Nanchang University, 999 Xuefu Avenue, Nanchang 330031 (China)

    2011-12-15

    Electrospun poly-({epsilon}-caprolactone) (PCL) nanofibers has been widely used in the medical prosthesis. However, poor hydrophilicity and the lack of natural recognition sites for covalent cell-recognition signal molecules to promote cell attachment have limited its utility as tissue scaffolds. In this study, Bilayer porous scaffolds based on PCL electrospun membranes and gelatin (GE) sponges were fabricated through soft hydrolysis of PCL electrospun followed by grafting gelatin onto the fiber surface, through crosslinking and freeze drying treatment of additional gelatin coat and grafted gelatin surface. GE sponges were stably anchored on PCL membrane surface with the aid of grafted GE molecules. The morphologies of bilayer porous scaffolds were observed through SEM. The contact angle of the scaffolds was 0 Degree-Sign , the mechanical properties of scaffolds were measured by tensile test, Young's moduli of PCL scaffolds before and after hydrolysis are 66-77.3 MPa and 62.3-75.4 MPa, respectively. Thus, the bilayer porous scaffolds showed excellent hydrophilic surface and desirable mechanical strength due to the soft hydrolysis and GE coat. The cell culture results showed that the adipose derived mesenchymal stem cells did more favor to adhere and grow on the bilayer porous scaffolds than on PCL electrospun membranes. The better cell affinity of the final bilayer scaffolds not only attributed to the surface chemistry but also the introduction of bilayer porous structure.

  20. Defensins promote fusion and lysis of negatively charged membranes.

    OpenAIRE

    Fujii, G; Selsted, M E; Eisenberg, D.

    1993-01-01

    Defensins, a family of cationic peptides isolated from mammalian granulocytes and believed to permeabilize membranes, were tested for their ability to cause fusion and lysis of liposomes. Unlike alpha-helical peptides whose lytic effects have been extensively studied, the defensins consist primarily of beta-sheet. Defensins fuse and lyse negatively charged liposomes but display reduced activity with neutral liposomes. These and other experiments suggest that fusion and lysis is mediated prima...

  1. Bilayer registry in a multicomponent asymmetric membrane: Dependence on lipid composition and chain length

    International Nuclear Information System (INIS)

    A question of considerable interest to cell membrane biology is whether phase segregated domains across an asymmetric bilayer are strongly correlated with each other and whether phase segregation in one leaflet can induce segregation in the other. We answer both these questions in the affirmative, using an atomistic molecular dynamics simulation to study the equilibrium statistical properties of a 3-component asymmetric lipid bilayer comprising an unsaturated palmitoyl-oleoyl-phosphatidyl-choline, a saturated sphingomyelin, and cholesterol with different composition ratios. Our simulations are done by fixing the composition of the upper leaflet to be at the coexistence of the liquid ordered (lo)-liquid disordered (ld) phases, while the composition of the lower leaflet is varied from the phase coexistence regime to the mixed ld phase, across a first-order phase boundary. In the regime of phase coexistence in each leaflet, we find strong transbilayer correlations of the lo domains across the two leaflets, resulting in bilayer registry. This transbilayer correlation depends sensitively upon the chain length of the participating lipids and possibly other features of lipid chemistry, such as degree of saturation. We find that the lo domains in the upper leaflet can induce phase segregation in the lower leaflet, when the latter is nominally in the mixed (ld) phase

  2. Bilayer registry in a multicomponent asymmetric membrane: Dependence on lipid composition and chain length

    Energy Technology Data Exchange (ETDEWEB)

    Polley, Anirban [Raman Research Institute, C.V. Raman Avenue, Bangalore 560080 (India); Mayor, Satyajit [National Centre for Biological Sciences (TIFR), Bellary Road, Bangalore 560065 (India); Rao, Madan, E-mail: madan@rri.res.in, E-mail: madan@ncbs.res.in [Raman Research Institute, C.V. Raman Avenue, Bangalore 560080 (India); National Centre for Biological Sciences (TIFR), Bellary Road, Bangalore 560065 (India)

    2014-08-14

    A question of considerable interest to cell membrane biology is whether phase segregated domains across an asymmetric bilayer are strongly correlated with each other and whether phase segregation in one leaflet can induce segregation in the other. We answer both these questions in the affirmative, using an atomistic molecular dynamics simulation to study the equilibrium statistical properties of a 3-component asymmetric lipid bilayer comprising an unsaturated palmitoyl-oleoyl-phosphatidyl-choline, a saturated sphingomyelin, and cholesterol with different composition ratios. Our simulations are done by fixing the composition of the upper leaflet to be at the coexistence of the liquid ordered (l{sub o})-liquid disordered (l{sub d}) phases, while the composition of the lower leaflet is varied from the phase coexistence regime to the mixed l{sub d} phase, across a first-order phase boundary. In the regime of phase coexistence in each leaflet, we find strong transbilayer correlations of the l{sub o} domains across the two leaflets, resulting in bilayer registry. This transbilayer correlation depends sensitively upon the chain length of the participating lipids and possibly other features of lipid chemistry, such as degree of saturation. We find that the l{sub o} domains in the upper leaflet can induce phase segregation in the lower leaflet, when the latter is nominally in the mixed (l{sub d}) phase.

  3. Alginate/chitosan based bi-layer composite membrane as potential sustained-release wound dressing containing ciprofloxacin hydrochloride

    Science.gov (United States)

    Han, Fei; Dong, Yang; Song, Aihua; Yin, Ran; Li, Sanming

    2014-08-01

    The aims of this research were to develop and evaluate a novel ciprofloxacin hydrochloride loaded bi-layer composite membrane based on alginate and chitosan. In vitro antimicrobial activity, drug permeation study, morphology, cytotoxicity, primary skin irritation and in vivo pharmacodynamics were investigated. Results showed that the membranes could inhibit the growth of microorganisms for longer than 7 days. And there was no significant decrease in the metabolic activity of the Hacat fibroblasts cells were treated with the membranes. No edema and erythema were observed after administration of membranes on the rabbit skin after 14 days. Moreover, the results of pharmacodynamics showed that the membranes were more effective in improving the wound healing process. In conclusion, a novel bi-layer composite membrane was developed and results suggested that it could be exploited as sustained-release wound dressings.

  4. Triglyceride Blisters in Lipid Bilayers: Implications for Lipid Droplet Biogenesis and the Mobile Lipid Signal in Cancer Cell Membranes

    DEFF Research Database (Denmark)

    Khandelia, Himanshu; Duelund, Lars; Pakkanen, Kirsi Inkeri; Ipsen, John Hjort

    2010-01-01

    aggregates of unknown function present in malignant cells, and to the early biogenesis of lipid droplets accommodated between the two leaflets of the endoplasmic reticulum membrane. The TO aggregates give the bilayer a blister-like appearance, and will hinder the formation of multi-lamellar phases in model......, and possibly living membranes. The blisters will result in anomalous membrane probe partitioning, which should be accounted for in the interpretation of probe-related measurements....

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

    Science.gov (United States)

    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. PMID:25845029

  6. Flavivirus cell entry and membrane fusion

    NARCIS (Netherlands)

    Smit, Jolanda M.; Moesker, Bastiaan; Rodenhuis-Zybert, Izabela; Wilschut, Jan

    2011-01-01

    Flaviviruses, such as dengue virus and West Nile virus, are enveloped viruses that infect cells through receptor-mediated endocytosis and fusion from within acidic endosomes. The cell entry process of flaviviruses is mediated by the viral E glycoprotein. This short review will address recent advance

  7. Chemotherapy Drugs Thiocolchicoside and Taxol Permeabilize Lipid Bilayer Membranes by Forming Ion Pores

    Science.gov (United States)

    Ashrafuzzaman, Md; Duszyk, M.; Tuszynski, J. A.

    2011-12-01

    We report ion channel formation by chemotherapy drugs: thiocolchicoside (TCC) and taxol (TXL) which primarily target tubulin but not only. For example, TCC has been shown to interact with GABAA, nuclear envelope and strychnine-sensitive glycine receptors. TXL interferes with the normal breakdown of microtubules inducing mitotic block and apoptosis. It also interacts with mitochondria and found significant chemotherapeutic applications for breast, ovarian and lung cancer. In order to better understand the mechanisms of TCC and TXL actions, we examined their effects on phospholipid bilayer membranes. Our electrophysiological recordings across membranes constructed in NaCl aqueous phases consisting of TCC or TXL under the influence of an applied transmembrane potential (V) indicate that both molecules induce stable ion flowing pores/channels in membranes. Their discrete current versus time plots exhibit triangular shapes which is consistent with a spontaneous time-dependent change of the pore conductance in contrast to rectangular conductance events usually induced by ion channels. These events exhibit conductance (~0.01-0.1 pA/mV) and lifetimes (~5-30 ms) within the ranges observed in e.g., gramicidin A and alamethicin channels. The channel formation probability increases linearly with TCC/TXL concentration and V and is not affected by pH (5.7 - 8.4). A theoretical explanation on the causes of chemotherapy drug induced ion pore formation and the pore stability has also been found using our recently discovered binding energy between lipid bilayer and the bilayer embedded ion channels using gramicidin A channels as tools. This picture of energetics suggests that as the channel forming agents approach to the lipids on bilayer the localized charge properties in the constituents of both channel forming agents (e.g., chemotherapy drugs in this study) and the lipids determine the electrostatic drug-lipid coupling energy through screened Coulomb interactions between the drug

  8. Chemotherapy Drugs Thiocolchicoside and Taxol Permeabilize Lipid Bilayer Membranes by Forming Ion Pores

    International Nuclear Information System (INIS)

    We report ion channel formation by chemotherapy drugs: thiocolchicoside (TCC) and taxol (TXL) which primarily target tubulin but not only. For example, TCC has been shown to interact with GABAA, nuclear envelope and strychnine-sensitive glycine receptors. TXL interferes with the normal breakdown of microtubules inducing mitotic block and apoptosis. It also interacts with mitochondria and found significant chemotherapeutic applications for breast, ovarian and lung cancer. In order to better understand the mechanisms of TCC and TXL actions, we examined their effects on phospholipid bilayer membranes. Our electrophysiological recordings across membranes constructed in NaCl aqueous phases consisting of TCC or TXL under the influence of an applied transmembrane potential (V) indicate that both molecules induce stable ion flowing pores/channels in membranes. Their discrete current versus time plots exhibit triangular shapes which is consistent with a spontaneous time-dependent change of the pore conductance in contrast to rectangular conductance events usually induced by ion channels. These events exhibit conductance (∼0.01-0.1 pA/mV) and lifetimes (∼5-30 ms) within the ranges observed in e.g., gramicidin A and alamethicin channels. The channel formation probability increases linearly with TCC/TXL concentration and V and is not affected by pH (5.7 - 8.4). A theoretical explanation on the causes of chemotherapy drug induced ion pore formation and the pore stability has also been found using our recently discovered binding energy between lipid bilayer and the bilayer embedded ion channels using gramicidin A channels as tools. This picture of energetics suggests that as the channel forming agents approach to the lipids on bilayer the localized charge properties in the constituents of both channel forming agents (e.g., chemotherapy drugs in this study) and the lipids determine the electrostatic drug-lipid coupling energy through screened Coulomb interactions between the

  9. Self-assembly of azobenzene bilayer membranes in binary ionic liquid-water nanostructured media.

    Science.gov (United States)

    Kang, Tejwant Singh; Ishiba, Keita; Morikawa, Masa-aki; Kimizuka, Nobuo

    2014-03-11

    Anionic azobenzene-containing amphiphile 1 (sodium 4-[4-(N-methyl-N-dodecylamino)phenylazo]benzenesulfonate) forms ordered bilayer membranes in binary ionic liquid (1-ethyl-3-methylimidazolium ethyl sulfate, [C2mim][C2OSO3])-water mixtures. The binary [C2mim][C2OSO3]-water mixture is macroscopically homogeneous at any mixing ratio; however, it possesses fluctuating nanodomains of [C2mim][C2OSO3] molecules as observed by dynamic light scattering (DLS). These nanodomains show reversible heat-induced mixing behavior with water. Although the amphiphile 1 is substantially insoluble in pure water, it is dispersible in the [C2mim][C2OSO3]-water mixtures. The concentration of [C2mim][C2OSO3] and temperature exert significant influences on the self-assembling characteristics of 1 in the binary media, as shown by DLS, transmission electron microscopy (TEM), UV-vis spectroscopy, and zeta-potential measurements. Bilayer membranes with rod- or dotlike nanostructures were formed at a lower content of [C2mim][C2OSO3] (2-30 v/v %), in which azobenzene chromophores adopt parallel molecular orientation regardless of temperature. In contrast, when the content of [C2mim][C2OSO3] is increased above 60 v/v %, azobenzene bilayers showed thermally reversible gel-to-liquid crystalline phase transition. The self-assembly of azobenzene amphiphiles is tunable depending on the volume fraction of [C2mim][C2OSO3] and temperature, which are associated with the solvation by nanoclusters in the binary [C2mim][C2OSO3]-water media. These observations clearly indicate that mixtures of water-soluble ionic liquids and water provide unique and valiant environments for ordered molecular self-assembly. PMID:24528277

  10. Comparing ion conductance recordings of synthetic lipid bilayers with cell membranes containing TRP channels

    CERN Document Server

    Laub, Katrine R; Blicher, Andreas; Madsen, Soren B; Luckhoff, Andreas; Heimburg, Thomas

    2011-01-01

    In this article we compare electrical conductance events from single channel recordings of three TRP channel proteins (TRPA1, TRPM2 and TRPM8) expressed in human embryonic kidney cells with channel events recorded on synthetic lipid membranes close to melting transitions. Ion channels from the TRP family are involved in a variety of sensory processes including thermo- and mechano-reception. Synthetic lipid membranes close to phase transitions display channel-like events that respond to stimuli related to changes in intensive thermodynamic variables such as pressure and temperature. TRP channel activity is characterized by typical patterns of current events dependent on the type of protein expressed. Synthetic lipid bilayers show a wide spectrum of electrical phenomena that are considered typical for the activity of protein ion channels. We find unitary currents, burst behavior, flickering, multistep-conductances, and spikes behavior in both preparations. Moreover, we report conductances and lifetimes for lipi...

  11. Fibrillation of β amyloid peptides in the presence of phospholipid bilayers and the consequent membrane disruption.

    Science.gov (United States)

    Qiang, Wei; Yau, Wai-Ming; Schulte, Jürgen

    2015-01-01

    Fibrillation of β amyloid (Aβ) peptides and the accumulation of amyloid plaques are considered as an important clinical hallmark to identify Alzheimer's disease (AD). The physiological connection between Aβ plaques and the disruption of neuronal cells has not been clearly understood. One hypothesis to explain the Aβ neurotoxicity is that the fibrillation process induces disruption to the cellular membrane. We studied the Aβ fibrillation process in two biologically relevant conditions with the peptide either pre-incorporated into or externally added to the synthetic phospholipid bilayers. These two sample preparation conditions mimic the physiological membrane proximities of Aβ peptides before and after the enzymatic cleavage of amyloid precursor protein (APP). Using thioflavin T (ThT) fluorescence and transmission electron microscopy (TEM), we were able to monitor the kinetics and morphological evolution of fibril formation, which was highly sensitive to the two sample preparation protocols. While the external addition protocol generates long and mature fibrils through normal fibrillation process, the pre-incubation protocol was found to stabilize the immature protofibrils. Fluorescence spectroscopy studies with doubly-labeled phospholipids indicated that there may be a lipid uptake process associated with the fibril formation. Solid state nuclear magnetic resonance (NMR) spectroscopy provided evidence for high resolution structural variations in fibrils formed with different protocols, and in particular the stabilization of long-range contact between N- and C-terminal β strands. In addition, disruption of phospholipid bilayers was supported by measurements with ³¹P chemical shifts and relaxation time constants. PMID:24769158

  12. The effect of acclimation temperature on the fusion kinetics of lipid vesicles derived from endoplasmic reticulum membranes of rainbow trout (Oncorhynchus mykiss) liver.

    Science.gov (United States)

    Miranda, Estuardo J; Hazel, Jeffrey R

    2002-02-01

    Membrane fusion is an obligatory step in many vital cellular processes. The well-established enrichment of bilayer-destabilizing lipids in membranes of poikilotherms subjected to growth at low temperatures leads to the prediction that such membranes will possess a greater propensity to undergo fusion. This hypothesis was explicitly tested in the present study by determining the kinetics of fusion between small unilamellar vesicles (SUVs) prepared from endoplasmic reticulum (ER) membranes of thermally-acclimated (to 5 and 20 degrees C) rainbow trout (Oncorhynchus mykiss) liver and bovine brain phosphatidylserine (BBPS). At temperatures above 10 degrees C, ER vesicles from 5 degrees C-acclimated trout, fused more rapidly and to a greater extent with BBPS vesicles (by average factors of 1.25- and 1.45-fold, respectively) than ER vesicles of 20 degrees C-acclimated trout. At temperatures >35 degrees C, apparent fusion rates declined while the extent of fusion increased in both acclimation groups. Fusion kinetics were found to be well correlated with and limited by the physical properties and phase state of the BBPS vesicles. These results indicate that dynamic attributes of biological membranes, such as the propensity to undergo fusion, are of potential regulatory significance and are partially conserved when growth or environmental temperature changes. PMID:11818217

  13. Membrane Bending Energy and Fusion Pore Kinetics in Ca2+-Triggered Exocytosis

    OpenAIRE

    Zhang, Zhen; Jackson, Meyer B.

    2010-01-01

    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 Ca2+-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...

  14. HIV fusion peptide penetrates, disorders, and softens T-cell membrane mimics.

    Science.gov (United States)

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

    2010-09-10

    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 K(C), a measure of membrane stiffness, and wide-angle x-ray scattering yielded the S(xray) orientational order parameter. Both FP23 and FPtri decreased K(C) and S(xray) 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

  15. Importin β Negatively Regulates Nuclear Membrane Fusion and Nuclear Pore Complex Assembly

    OpenAIRE

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

    2003-01-01

    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 β negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin β is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin β down-regulation of membrane fusion is Ran-GTP reversible. Inde...

  16. Membrane interactions of ternary phospholipid/cholesterol bilayers and encapsulation efficiencies of a RIP II protein.

    Science.gov (United States)

    Manojlovic, V; Winkler, K; Bunjes, V; Neub, A; Schubert, R; Bugarski, B; Leneweit, G

    2008-07-15

    Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P(min), the bilayer lysis tensions gamma(l) were determined. The increase in P(min) and gamma(l) with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron

  17. Tethered bilayer membranes as a complementary tool for functional and structural studies: The pyolysin case.

    Science.gov (United States)

    Preta, Giulio; Jankunec, Marija; Heinrich, Frank; Griffin, Sholeem; Sheldon, Iain Martin; Valincius, Gintaras

    2016-09-01

    We demonstrate the use of tethered bilayer lipid membranes (tBLMs) as an experimental platform for functional and structural studies of membrane associated proteins by electrochemical techniques. The reconstitution of the cholesterol-dependent cytolysin (CDC) pyolysin (PLO) from Trueperella pyogenes into tBLMs was followed in real-time by electrochemical impedance spectroscopy (EIS). Changes of the EIS parameters of the tBLMs upon exposure to PLO solutions were consistent with the dielectric barrier damage occurring through the formation of water-filled pores in membranes. Parallel experiments involving a mutant version of PLO, which is able to bind to the membranes but does not form oligomer pores, strengthen the reliability of this methodology, since no change in the electrochemical impedance was observed. Complementary atomic force microscopy (AFM) and neutron reflectometry (NR) measurements revealed structural details of the membrane bound PLO, consistent with the structural transformations of the membrane bound toxins found for other cholesterol dependent cytolysins. In this work, using the tBLMs platform we also observed a protective effect of the dynamin inhibitor Dynasore against pyolysin as well as pneumolysin. An effect of Dynasore in tBLMs, which was earlier observed in experiments with live cells, confirms the biological relevance of the tBLMs models, as well as demonstrates the potential of the electrochemical impedance spectroscopy to quantify membrane damage by the pore forming toxins. In conclusion, tBLMs are a reliable and complementary method to explore the activity of CDCs in eukaryotic cells and to develop strategies to limit the toxic effects of CDCs. PMID:27211243

  18. Oriented Circular Dichroism: A Method to Characterize Membrane-Active Peptides in Oriented Lipid Bilayers.

    Science.gov (United States)

    Bürck, Jochen; Wadhwani, Parvesh; Fanghänel, Susanne; Ulrich, Anne S

    2016-02-16

    The structures of membrane-bound polypeptides are intimately related to their functions and may change dramatically with the lipid environment. Circular dichroism (CD) is a rapid analytical method that requires relatively low amounts of material and no labeling. Conventional CD is routinely used to monitor the secondary structure of peptides and proteins in solution, for example, in the presence of ligands and other binding partners. In the case of membrane-active peptides and transmembrane proteins, these measurements can be applied to, and remain limited to, samples containing detergent micelles or small sonicated lipid vesicles. Such traditional CD analysis reveals only secondary structures. With the help of an oriented circular dichroism (OCD) setup, however, based on the preparation of macroscopically oriented lipid bilayers, it is possible to address the membrane alignment of a peptide in addition to its conformation. This approach has been mostly used for α-helical peptides so far, but other structural elements are conceivable as well. OCD analysis relies on Moffitt's theory, which predicts that the electronic transition dipole moments of the backbone amide bonds in helical polypeptides are polarized either parallel or perpendicular to the helix axis. The interaction of the electric field vector of the circularly polarized light with these transitions results in an OCD spectrum of a membrane-bound α-helical peptide, which exhibits a characteristic line shape and reflects the angle between the helix axis and the bilayer normal. For parallel alignment of a peptide helix with respect to the membrane surface (S-state), the corresponding "fingerprint" CD band around 208 nm will exhibit maximum negative amplitude. If the helix changes its alignment via an obliquely tilted (T-state) to a fully inserted transmembrane orientation (I-state), the ellipticity at 208 nm decreases and the value approaches zero due to the decreased interactions between the field and the

  19. Tetra- and Penta-Cyclic Triterpenes Interaction with Lipid Bilayer Membrane: A Structural Comparative Study.

    Science.gov (United States)

    Abboud, Rola; Charcosset, Catherine; Greige-Gerges, Hélène

    2016-06-01

    The effect of tetracyclic (cortisol, prednisolone, and 9-fluorocortisol acetate) and pentacyclic (uvaol and erythrodiol) triterpenes (TTPs) on the fluidity of dipalmitoyl phosphatidyl choline (DPPC) liposome membrane was investigated by differential scanning calorimetry, Raman spectroscopy, and fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Liposomes were prepared in the absence and presence of TTPs at molar ratios DPPC:TTP 100:1, 100:2.5, and 100:10. All the studied TTPs abolished the pre-transition and modified the intensity of the Raman peak at 715 cm(-1) proving the interaction of TTP molecules with the choline head group of phospholipids. An increase in the Raman height intensity ratios of the peaks I 2935/2880, I 2844/2880, and I 1090/1130, giving information about the ratio disorder/order of the alkyl chains, and a decrease of the main transition temperature demonstrated the interaction of TTPs with the alkyl chains. The tetracyclic TTPs produced broadening of the phase transition profile. Besides, a scarcely splitting of the main transition peak was obtained with prednisolone and 9-fluorocortisol acetate. The results of fluorescence depolarization of DPH showed that the studied molecules fluidized the liposomal membrane at 25, 41, and 50 °C. Pentacyclic TTPs, being more hydrophobic than tetracyclic ones, demonstrated higher fluidizing effect than tetracyclic TTPs in the liquid crystalline phase suggesting a deeper incorporation in the lipid bilayer. The presence of a free polar head group at the ring D seems to control the TTP incorporation in the bilayer and consequently its effect on the membrane fluidity. PMID:26759229

  20. Study of the ion-channel behavior on glassy carbon electrode supported bilayer lipid membranes stimulated by perchlorate anion

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Zhiquan; Shi, Jun; Huang, Weimin, E-mail: huangwm@jlu.edu.cn

    2015-10-01

    In this paper, a kind of didodecyldimethylammonium bromide (DDAB) layer membranes was supported on a glassy carbon electrode (GCE). We studied the ion channel behavior of the supported bilayer lipid membrane by scanning electrochemical microscopy (SCEM) in tris(2,2′-bipyridine) ruthenium(II) solution. Perchlorate anion was used as a presence of stimulus and ruthenium(II) complex cations as the probing ions for the measurement of SECM, the lipid membrane channel was opened and exhibited the behavior of distinct SECM positive feedback curve. The channel was in a closed state in the absence of perchlorate anions while reflected the behavior of SECM negative feedback curve. The rates of electron transfer reaction in the lipid membranes surface were detected and it was dependant on the potential of SECM. - Highlights: • The rates of electron transfer reaction in the lipid membranes surface were detected. • Dynamic investigations of ion-channel behavior of supported bilayer lipid membranes by scanning electrochemical microscopy • A novel way to explore the interaction between molecules and supported bilayer lipid membranes.

  1. Study of the ion-channel behavior on glassy carbon electrode supported bilayer lipid membranes stimulated by perchlorate anion

    International Nuclear Information System (INIS)

    In this paper, a kind of didodecyldimethylammonium bromide (DDAB) layer membranes was supported on a glassy carbon electrode (GCE). We studied the ion channel behavior of the supported bilayer lipid membrane by scanning electrochemical microscopy (SCEM) in tris(2,2′-bipyridine) ruthenium(II) solution. Perchlorate anion was used as a presence of stimulus and ruthenium(II) complex cations as the probing ions for the measurement of SECM, the lipid membrane channel was opened and exhibited the behavior of distinct SECM positive feedback curve. The channel was in a closed state in the absence of perchlorate anions while reflected the behavior of SECM negative feedback curve. The rates of electron transfer reaction in the lipid membranes surface were detected and it was dependant on the potential of SECM. - Highlights: • The rates of electron transfer reaction in the lipid membranes surface were detected. • Dynamic investigations of ion-channel behavior of supported bilayer lipid membranes by scanning electrochemical microscopy • A novel way to explore the interaction between molecules and supported bilayer lipid membranes

  2. Thermodynamic free energy methods to investigate shape transitions in bilayer membranes

    Science.gov (United States)

    Ramakrishnan, N.; Tourdot, Richard W.

    2016-01-01

    The conformational free energy landscape of a system is a fundamental thermodynamic quantity of importance particularly in the study of soft matter and biological systems, in which the entropic contributions play a dominant role. While computational methods to delineate the free energy landscape are routinely used to analyze the relative stability of conformational states, to determine phase boundaries, and to compute ligand-receptor binding energies its use in problems involving the cell membrane is limited. Here, we present an overview of four different free energy methods to study morphological transitions in bilayer membranes, induced either by the action of curvature remodeling proteins or due to the application of external forces. Using a triangulated surface as a model for the cell membrane and using the framework of dynamical triangulation Monte Carlo, we have focused on the methods of Widom insertion, thermodynamic integration, Bennett acceptance scheme, and umbrella sampling and weighted histogram analysis. We have demonstrated how these methods can be employed in a variety of problems involving the cell membrane. Specifically, we have shown that the chemical potential, computed using Widom insertion, and the relative free energies, computed using thermodynamic integration and Bennett acceptance method, are excellent measures to study the transition from curvature sensing to curvature inducing behavior of membrane associated proteins. The umbrella sampling and WHAM analysis has been used to study the thermodynamics of tether formation in cell membranes and the quantitative predictions of the computational model are in excellent agreement with experimental measurements. Furthermore, we also present a method based on WHAM and thermodynamic integration to handle problems related to end-point-catastrophe that are common in most free energy methods.

  3. On the freezing behavior and diffusion of water in proximity to single-supported zwitterionic and anionic bilayer lipid membranes

    DEFF Research Database (Denmark)

    Miskowiec, A.; Buck, Z. N.; Brown, M. C.;

    2014-01-01

    We compare the freezing/melting behavior of water hydrating single-supported bilayers of a zwitterionic lipid DMPC with that of an anionic lipid DMPG. For both membranes, the temperature dependence of the elastically scattered neutron intensity indicates distinct water types undergoing...

  4. Non-bilayer lipids are required for efficient protein transport across the plasma membrane of Escherichia coli.

    OpenAIRE

    Rietveld, A G; Koorengevel, M C; de Kruijff, B.

    1995-01-01

    The construction of a mutant Escherichia coli strain which cannot synthesize phosphatidylethanolamine provides a tool to study the involvement of non-bilayer lipids in membrane function. This strain produces phosphatidylglycerol and cardiolipin (CL) as major membrane constituents and requires millimolar concentrations of divalent cations for growth. In this strain, the lipid phase behaviour is tightly regulated by adjustment of the level of CL which favours a nonbilayer organization in the pr...

  5. Formation of Cholesterol Bilayer Domains Precedes Formation of Cholesterol Crystals in Cholesterol/Dimyristoylphosphatidylcholine Membranes: EPR and DSC Studies

    OpenAIRE

    Mainali, Laxman; Raguz, Marija; Subczynski, Witold K.

    2013-01-01

    Saturation-recovery EPR along with DSC were used to determine the cholesterol content at which pure cholesterol bilayer domains (CBDs) and cholesterol crystals begin to form in dimyristoylphosphatidylcholine (DMPC) membranes. To preserve compositional homogeneity throughout the membrane suspension, lipid multilamellar dispersions were prepared using a rapid solvent exchange method. The cholesterol content increased from 0 to 75 mol%. With spin-labeled cholesterol analogs it was shown that the...

  6. Particle-based simulations of bilayer membranes: self-assembly, structural analysis, and shock-wave damage

    Science.gov (United States)

    Steinhauser, Martin O.; Schindler, Tanja

    2016-08-01

    We report on the results of particle-based, coarse-grained molecular dynamics simulations of amphiphilic lipid molecules in aqueous environment where the membrane structures at equilibrium are subsequently exposed to strong shock waves, and their damage is analyzed. The lipid molecules self-assemble from unbiased random initial configurations to form stable bilayer membranes, including closed vesicles. During self-assembly of lipid molecules, we observe several stages of clustering, starting with many small clusters of lipids, gradually merging together to finally form one single bilayer membrane. We find that the clustering of lipids sensitively depends on the hydrophobic interaction h_c of the lipid tails in our model and on temperature T of the system. The self-assembled bilayer membranes are quantitatively analyzed at equilibrium with respect to their degree of order and their local structure. We also show that—by analyzing the membrane fluctuations and using a linearized theory— we obtain area compression moduli K_A and bending stiffnesses κ_B for our bilayer membranes which are within the experimental range of in vivo and in vitro measurements of biological membranes. We also discuss the density profile and the pair correlation function of our model membranes at equilibrium which has not been done in previous studies of particle-based membrane models. Furthermore, we present a detailed phase diagram of our lipid model that exhibits a sol-gel transition between quasi-solid and fluid domains, and domains where no self-assembly of lipids occurs. In addition, we present in the phase diagram the conditions for temperature T and hydrophobicity h_c of the lipid tails of our model to form closed vesicles. The stable bilayer membranes obtained at equilibrium are then subjected to strong shock waves in a shock tube setup, and we investigate the damage in the membranes due to their interaction with shock waves. Here, we find a transition from self

  7. Conflicting views on the membrane fusion machinery and the fusion pore

    DEFF Research Database (Denmark)

    Sørensen, Jakob B

    2009-01-01

    of the assembly of the fusogenic SNARE-complex. Here, I review conflicting views on the function of the core fusion machinery consisting of the SNAREs, Munc18, complexin, and synaptotagmin. Munc18 controls docking of vesicles to the plasma membrane and initial SNARE-complex assembly, whereas complexin...

  8. Tubular lipid membranes pulled from vesicles: Dependence of system equilibrium on lipid bilayer curvature

    Science.gov (United States)

    Golushko, I. Yu.; Rochal, S. B.

    2016-01-01

    Conditions of joint equilibrium and stability are derived for a spherical lipid vesicle and a tubular lipid membrane (TLM) pulled from this vesicle. The obtained equations establish relationships between the geometric and physical characteristics of the system and the external parameters, which have been found to be controllable in recent experiments. In particular, the proposed theory shows that, in addition to the pressure difference between internal and external regions of the system, the variable spontaneous average curvature of the lipid bilayer (forming the TLM) also influences the stability of the lipid tube. The conditions for stability of the cylindrical phase of TLMs after switching off the external force that initially formed the TLM from a vesicle are discussed. The loss of system stability under the action of a small axial force compressing the TLM is considered.

  9. Interactions between oxicams and membrane bilayers: an explanation for their different COX selectivity.

    Science.gov (United States)

    Lúcio, M; Ferreira, H; Lima, José L F C; Reis, Salette

    2006-09-01

    Meloxicam was launched as a major new NSAID for the treatment of arthritis following extensive published research confirming its selectivity for COX-2. Several studies proposed possible explanations for its effectiveness and superior safety profile. The proposed theories included chemical structural relationships between meloxicam and other effective NSAIDs with low gastrointestinal toxic effects. However, other oxicams have similar chemical groups, but despite this, are not considered COX-2 selective drugs and exhibit less gastric tolerance. Hence, the aim of this work was to investigate the interactions between oxicams and biomembrane models as it could influence their resorption from the upper gastrointestinal tract and may affect their local gastromucosal tolerability. The partition of oxicams within membranes was determined by calculating their partition coefficients between liposomes and water. Moreover, their location within the bilayer was determined by fluorescence quenching. Finally, zeta-potential measurements were made to complete the information about the binding behaviour of the oxicams and steady-state anisotropy measurements were made to determine their induced perturbation in membrane structure. These studies proved that, in spite of structural similarities, oxicams present different interactions with membranes making possible a virtual division of the class in two groups. Tenoxicam and piroxicam known as COX-1 inhibitors demonstrated higher partition capacity in liposomes/water systems together with a smaller ability to change the membrane fluidity and surface potential. In contrast lornoxicam and meloxicam, which demonstrated activity against COX-2, have revealed smaller partition capacity in liposomes/water systems together with a higher ability to change the membrane fluidity and surface potential. PMID:17017983

  10. Reversible Merger of Membranes at the Early Stage of Influenza Hemagglutinin-mediated Fusion

    OpenAIRE

    Leikina, Eugenia; Chernomordik, Leonid V.

    2000-01-01

    Fusion mediated by influenza hemagglutinin (HA), a prototype fusion protein, is commonly detected as lipid and content mixing between fusing cells. Decreasing the surface density of fusion-competent HA inhibited these advanced fusion phenotypes and allowed us to identify an early stage of fusion at physiological temperature. Although lipid flow between membranes was restricted, the contacting membrane monolayers were apparently transiently connected, as detected by the transformation of this ...

  11. The Gaussian Curvature Elastic Energy of Intermediates in Membrane Fusion

    OpenAIRE

    Siegel, David P.

    2008-01-01

    The Gaussian curvature elastic energy contribution to the energy of membrane fusion intermediates has usually been neglected because the Gaussian curvature elastic modulus, κ, was unknown. It is now possible to measure κ for phospholipids that form bicontinuous inverted cubic (QII) phases. Here, it is shown that one can estimate κ for lipids that do not form QII phases by studying the phase behavior of lipid mixtures. The method is used to estimate κ for several lipid compositions in excess w...

  12. Theory of electroporation of planar bilayer membranes: predictions of the aqueous area, change in capacitance, and pore-pore separation.

    OpenAIRE

    Freeman, S. A.; Wang, M. A.; Weaver, J C

    1994-01-01

    A large increase in the transmembrane voltage, U(t), of a fluid bilayer membrane is believed to result in the occurrence of temporary aqueous pathways ("pores") across the membrane. The number, size, and evolution dynamics of these pores are expected to be crucial to the transport of water-soluble species ranging from small ions to macromolecules such as proteins and nucleic acids. In this paper we use a transient aqueous pore theory to estimate the fraction of the membrane area, Fw(t), which...

  13. Electrically silent anion transport through bilayer lipid membrane induced by tributyltin and triethyllead.

    Science.gov (United States)

    Antonenko YuN

    1990-02-01

    The method of the measurement of the nonelectrogenic fluxes of hydrogen (or hydroxyl) ions (JH) based on the local proton gradients formation in the unstirred layers near a bilayer lipid membrane (BLM) is applied for recording the nonelectrogenic anion/OH- exchange on BLM induced by tributyltin (TBT) and a novel carrier (Hager, A., Moser, I., & Berthold, W. 1987. Z. Naturforsch., 42C:1116-1120), triethyllead (TEL). This method has been used previously for measuring the cation fluxes through BLM. TBT and TEL are shown to be equally efficient in the induction of Cl-/OH- exchange. JH induced by TBT is constant at 4 less than pH less than 7. JH decreases at pH less than 4 and pH greater than 7. Both ionophores have a transport sequence: I- greater than Br- greater than Cl- greater than F-. The quantitative measurements reveal that TEL better discriminates these four anions than TBT. It is concluded that this method may prove helpful in a search and study of anion/OH(-)-exchangers isolated from natural membranes. PMID:2319590

  14. Diffusion studies on permeable nitroxyl spin probes through bilayer lipid membranes: A low frequency ESR study

    Science.gov (United States)

    Meenakumari, V.; Utsumi, Hideo; Ichikawa, Kazuhiro; Yamada, Ken-ichi; Hyodo, Fuminori; Jawahar, A.; Benial, A. Milton Franklin

    2015-06-01

    Electron spin resonance (ESR) studies were carried out for permeable 2mM 14N-labeled deutrated 3 Methoxy carbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) in pure water and 1mM, 2mM, 3mM, 4mM concentration of 14N-labeled deutrated MC-PROXYL in 400mM concentration of liposomal solution by using a 300 MHz ESR spectrometer. The ESR parameters such as linewidth, hyperfine coupling constant, g-factor, partition parameter and permeability were reported for these samples. The line broadening was observed for the nitroxyl spin probe in the liposomal solution. The line broadening indicates that the high viscous nature of the liposomal solution. The partition parameter and permeability values indicate the maximum diffusion of nitroxyl spin probes in the bilayer lipid membranes at 2 mM concentration of nitroxyl radical. This study illustrates that ESR can be used to differentiate between the intra and extra- membrane water by loading the liposome vesicles with a lipid-permeable nitroxyl spin probe. From the ESR results, the spin probe concentration was optimized as 2mM in liposomal solution for ESR phantom studies/imaging, invivo and invitro experiments.

  15. Diffusion studies on permeable nitroxyl spin probes through bilayer lipid membranes: A low frequency ESR study

    Energy Technology Data Exchange (ETDEWEB)

    Meenakumari, V.; Benial, A. Milton Franklin, E-mail: miltonfranklin@yahoo.com [Department of Physics, NMSSVN College, Nagamalai, Madurai-625019, Tamilnadu (India); Utsumi, Hideo; Ichikawa, Kazuhiro; Yamada, Ken-ichi [Department of Bio-functional Science, Kyushu University, Fukuoka (Japan); Hyodo, Fuminori [Innovation Center for Medical Redox Navigation, Kyushu University, Fukuoka (Japan); Jawahar, A. [Department of Chemistry, NMSSVN College, Nagamalai, Madurai-625019, Tamilnadu (India)

    2015-06-24

    Electron spin resonance (ESR) studies were carried out for permeable 2mM {sup 14}N-labeled deutrated 3 Methoxy carbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) in pure water and 1mM, 2mM, 3mM, 4mM concentration of 14N-labeled deutrated MC-PROXYL in 400mM concentration of liposomal solution by using a 300 MHz ESR spectrometer. The ESR parameters such as linewidth, hyperfine coupling constant, g-factor, partition parameter and permeability were reported for these samples. The line broadening was observed for the nitroxyl spin probe in the liposomal solution. The line broadening indicates that the high viscous nature of the liposomal solution. The partition parameter and permeability values indicate the maximum diffusion of nitroxyl spin probes in the bilayer lipid membranes at 2 mM concentration of nitroxyl radical. This study illustrates that ESR can be used to differentiate between the intra and extra- membrane water by loading the liposome vesicles with a lipid-permeable nitroxyl spin probe. From the ESR results, the spin probe concentration was optimized as 2mM in liposomal solution for ESR phantom studies/imaging, invivo and invitro experiments.

  16. Monitoring of cholesterol oxidation in a lipid bilayer membrane using streptolysin O as a sensing and signal transduction element.

    Science.gov (United States)

    Shoji, Atsushi; Ikeya, Kana; Aoyagi, Miki; Takatsuji, Ryutaro; Yanagida, Akio; Shibusawa, Yoichi; Sugawara, Masao

    2016-09-01

    Streptolysin O (SLO), which recognizes sterols and forms nanopores in lipid membranes, is proposed as a sensing element for monitoring cholesterol oxidation in a lipid bilayer. The structural requirements of eight sterols for forming nanopores by SLO confirmed that a free 3-OH group in the β-configuration of sterols is required for recognition by SLO in a lipid bilayer. The extent of nanopore formation by SLO in lipid bilayers increased in the order of cholestanolcholesterolcholesterol and in a sterol concentration-dependent manner. The immobilization of liposomes consisting of PC, cholesterol and 4-cholesten-3-one exhibited a linear relationship between calcein permeability and the molar ratio of cholesterol and 4-cholesten-3-one. The SLO-based method was successfully applied for monitoring of cholesterol oxidase-mediated oxidation of cholesterol in a lipid bilayer. The potential of the SLO nanopore-based method for monitoring cholesterol oxidation in a lipid bilayer by other oxidative enzymes is also discussed. PMID:27362457

  17. Membrane interaction and structure of the transmembrane domain of influenza hemagglutinin and its fusion peptide complex

    Directory of Open Access Journals (Sweden)

    Lin Chi-Hui

    2008-01-01

    Full Text Available Abstract Background To study the organization and interaction with the fusion domain (or fusion peptide, FP of the transmembrane domain (TMD of influenza virus envelope glycoprotein for its role in membrane fusion which is also essential in the cellular trafficking of biomolecules and sperm-egg fusion. Results The fluorescence and gel electrophoresis experiments revealed a tight self-assembly of TMD in the model membrane. A weak but non-random interaction between TMD and FP in the membrane was found. In the complex, the central TMD oligomer was packed by FP in an antiparallel fashion. FP insertion into the membrane was altered by binding to TMD. An infrared study exhibited an enhanced membrane perturbation by the complex formation. A model was built to illustrate the role of TMD in the late stages of influenza virus-mediated membrane fusion reaction. Conclusion The TMD oligomer anchors the fusion protein in the membrane with minimal destabilization to the membrane. Upon associating with FP, the complex exerts a synergistic effect on the membrane perturbation. This effect is likely to contribute to the complete membrane fusion during the late phase of fusion protein-induced fusion cascade. The results presented in the work characterize the nature of the interaction of TMD with the membrane and TMD in a complex with FP in the steps leading to pore initiation and dilation during virus-induced fusion. Our data and proposed fusion model highlight the key role of TMD-FP interaction and have implications on the fusion reaction mediated by other type I viral fusion proteins. Understanding the molecular mechanism of membrane fusion may assist in the design of anti-viral drugs.

  18. Viral membrane fusion: is glycoprotein G of rhabdoviruses a representative of a new class of viral fusion proteins?

    Directory of Open Access Journals (Sweden)

    A.T. Da Poian

    2005-06-01

    Full Text Available Enveloped viruses always gain entry into the cytoplasm by fusion of their lipid envelope with a cell membrane. Some enveloped viruses fuse directly with the host cell plasma membrane after virus binding to the cell receptor. Other enveloped viruses enter the cells by the endocytic pathway, and fusion depends on the acidification of the endosomal compartment. In both cases, virus-induced membrane fusion is triggered by conformational changes in viral envelope glycoproteins. Two different classes of viral fusion proteins have been described on the basis of their molecular architecture. Several structural data permitted the elucidation of the mechanisms of membrane fusion mediated by class I and class II fusion proteins. In this article, we review a number of results obtained by our laboratory and by others that suggest that the mechanisms involved in rhabdovirus fusion are different from those used by the two well-studied classes of viral glycoproteins. We focus our discussion on the electrostatic nature of virus binding and interaction with membranes, especially through phosphatidylserine, and on the reversibility of the conformational changes of the rhabdovirus glycoprotein involved in fusion. Taken together, these data suggest the existence of a third class of fusion proteins and support the idea that new insights should emerge from studies of membrane fusion mediated by the G protein of rhabdoviruses. In particular, the elucidation of the three-dimensional structure of the G protein or even of the fusion peptide at different pH's might provide valuable information for understanding the fusion mechanism of this new class of fusion proteins.

  19. Enhanced oxygen separation through robust freeze-cast bilayered dual-phase membranes.

    Science.gov (United States)

    Gaudillere, Cyril; Garcia-Fayos, Julio; Balaguer, María; Serra, José M

    2014-09-01

    Dual-phase oxygen-permeable asymmetric membranes with enhanced oxygen permeation were prepared by combining freeze-casting, screen-printing, and constraint-sintering techniques. The membranes were evaluated under oxyfuel operating conditions. The prepared membranes are composed of an original ice-templated La(0.6)Sr(0.4)Co(0.2)Fe(0.8)O(3-δ) support with hierarchically oriented porosity and a top fully densified bilayered coating comprising a 10 μm-thick La(0.6)Sr(0.4)Co(0.2)Fe(0.8)O(3-δ) layer and a top protective 8 μm-thick layer made of an optimized NiFe2O4/Ce(0.8)Tb(0.2)O(2-δ) composite synthesized by the one-pot Pechini method. Preliminary analysis confirmed the thermochemical compatibility of the three involved phases at high temperature without any additional phase detected. This membrane exhibited a promising oxygen permeation value of 4.8 mL min(-1)  cm(-2) at 1000 °C upon using Ar and air as the sweep and feed gases, respectively. Mimicking oxyfuel operating conditions by switching argon to pure CO2 as a sweep gas at 1000 °C and air as feed enabled an oxygen flux value of 5.6 mL min(-1)  cm(-2) to be reached. Finally, under the same conditions and increasing the oxygen partial pressure to 0.1 MPa in the feed, the oxygen permeation reached 12 mL min(-1)  cm(-2). The influence of CO2 content in the sweep gas was studied and its reversible and positive effect over oxygen permeation at temperatures equal to or above 950 °C was revealed. Finally, the membrane stability over a period of 150 h under CO2-rich sweep gas showed a low degradation rate of 2.4×10(-2)  mL min(-1)  cm(-2) per day. PMID:25070608

  20. Membrane technologies for tritium recovering in the fusion fuel cycle

    International Nuclear Information System (INIS)

    Palladium and palladium-silver permeators have been obtained by coating porous ceramic tubes with a thin metal layer. Three coating techniques have been studied and characterized: sputtering, chemical electroless deposition and cold-rolling. The Pd-Ag membranes obtained by cold-rolling and annealing of thin metal foils have shown complete hydrogen selectivity and chemical and physical stability meeting the requirements of the fuel cycle applications. These rolled membranes have been tested at 300-400 deg. C with a hydrogen transmembrane pressure in the range of 100-280 kPa and hydrogen flow rates up to 2.5x10-6m3/s. By filling the Pd-Ag membranes with a catalyst selective for the water gas shift reaction, membrane reactors have been obtained for recovering hydrogen isotopes in elemental form from tritiated water. Particularly, a closed-loop process based on a Pd-Ag membrane reactor has been studied for the tritium recovery system of an ITER scale fusion reactor. (author)

  1. An Ion Switch Regulates Fusion of Charged Membranes

    Science.gov (United States)

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

    2011-01-01

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

  2. Fusogenic segments of bovine leukemia virus and simian immunodeficiency virus are interchangeable and mediate fusion by means of oblique insertion in the lipid bilayer of their target cells.

    OpenAIRE

    Vonèche, V; Portetelle, D; Kettmann, R; Willems, L; Limbach, K.; E. Paoletti; Ruysschaert, J M; Burny, A; Brasseur, R.

    1992-01-01

    Modified bovine leukemia virus (BLV) glycoproteins were expressed by using vaccinia virus recombinants, and their fusogenic capacities were examined by a syncytia-formation assay. This analysis indicates that (i) both BLV envelope glycoproteins gp51 and gp30 are necessary for cell fusion; (ii) insertion of the N-terminal segment of gp30 (fusion peptide) into the lipid bilayer in an oblique orientation, as predicted by computer conformational analysis, results in fusogenic capacities higher th...

  3. A compensatory mutation provides resistance to disparate HIV fusion inhibitor peptides and enhances membrane fusion.

    Directory of Open Access Journals (Sweden)

    Matthew P Wood

    Full Text Available Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide, are peptides designed to competitively inhibit the viral fusion protein gp41. With the emergence of drug resistance, there is an increased need for effective and unique alternatives within this class of antivirals. One such alternative is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide due to its specific antiviral activity, lack of toxicity to cells and tissues, and safety in animals. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. In previous studies, we identified a mutant, R5-tropic virus that had evolved partial resistance to RC-101 during in vitro selection. Here, we report that a secondary mutation in gp41 was found to restore replicative fitness, membrane fusion, and the rate of viral entry, which were compromised by an initial mutation providing partial RC-101 resistance. Interestingly, we show that RC-101 is effective against two enfuvirtide-resistant mutants, demonstrating the clinical importance of RC-101 as a unique fusion inhibitor. These findings both expand our understanding of HIV drug-resistance to diverse peptide fusion inhibitors and emphasize the significance of compensatory gp41 mutations.

  4. Effect of increase in orientational order of lipid chains and head group spacing on non steroidal anti-inflammatory drug induced membrane fusion.

    Science.gov (United States)

    Roy, Sutapa Mondal; Bansode, Amol S; Sarkar, Munna

    2010-12-21

    Membrane fusion is a key event in many biological processes. The fusion process, both in vivo and in vitro, is induced by different agents which include mainly proteins and peptides. For protein- and peptide-mediated membrane fusion, conformational reorganization serves as a driving force. Small drug molecules do not share this advantage; hence, drug induced membrane fusion occurring in absence of any other fusogenic agent and at physiologically relevant concentration of the drugs is a very rare event. To date, only three drugs, namely, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx), belonging to the oxicam group of non steroidal anti-inflammatory drugs (NSAIDs), have been shown by us to induce fusion at very low drug to lipid ratio without the aid of any other fusogenic agent. In our continued effort to understand the interplay of different physical and chemical parameters of both the participating drugs and the membrane on the mechanism of this drug induced membrane fusion, we present here the effect of increase in orientational order of the lipid chains and increase in head group spacing. This is achieved by studying the effect of low concentration cholesterol (gel to fluid transition temperature, is mainly known to increase orientational order of the lipid chains and increase head group spacing. To isolate the effect of these parameters, small unilameller vesicles (SUVs) formed by dimyristoylphosphatidylcholine (DMPC) with an average diameter of 50-60 nm were used as simple model membranes. Fluorescence assays were used to probe the time dependence of lipid mixing, content mixing, and leakage and also used to determine the partitioning of the drugs in the membrane bilayer. Differential scanning calorimetry (DSC) was used to study the effect of drugs in the presence of cholesterol on the chain-melting temperature which reflects the fluidization effect of the hydrophobic tail region of the bilayer. Our results show contradictory effect of low concentration

  5. Reduction in lateral lipid mobility of lipid bilayer membrane by atmospheric pressure plasma irradiation

    Science.gov (United States)

    Suda, Yoshiyuki; Tero, Ryugo; Yamashita, Ryuma; Yusa, Kota; Takikawa, Hirofumi

    2016-03-01

    Plasma medicine is an emerging research field in which various applications of electrical discharge, especially in the form of nonequilibrium plasma at atmospheric pressure, are examined, for example, the application of plasma to biological targets for various purposes such as selective killing of tumor cells and blood stanching. We have focused on the behavior of an artificial cell membrane system at the solid-liquid interface. To evaluate the lateral lipid mobility, we measured the diffusion coefficient of the supported lipid bilayer (SLB) composed of dioleoylphosphatidylcholine with fluorescence recovery after photobleaching by confocal laser scanning microscopy. It was found that the diffusion coefficient was decreased by plasma irradiation and that the diffusion coefficient decreasing rate proceeded with increasing plasma power. We investigated the effects of stimulation with an equilibrium chemical, H2O2, on the SLB and confirmed that the diffusion coefficient did not change at least up to a H2O2 concentration of 5 mM. These results indicate that transient active species generated by plasma play critical roles in the reduction in SLB fluidity. The effects of the two generated major oxidized lipid species, hydroxyl- or hydroperoxy-phosphatidylcholine (PC) and acyl-chain-truncated PCs terminated with aldehyde or carboxyl group, on lateral lipid mobility are discussed.

  6. Study of the Ion Channel Behavior of Didodecyldimethylammonium Bromide Formed Bilayer Lipid Membrane Stimulated by PF-6

    Institute of Scientific and Technical Information of China (English)

    TONG,Yue-Hong; HAN,Xiao-Jun; WANG,Er-Kang

    2003-01-01

    Bilayer lipid membranes ( BLM ) formed from didodecyldimethylammonium bromide were made on the freshly exposed surface ofa glassy carbon (GC) ani were demonstrated by the ac impedance spectroscopy. The ion channels of membrane properties induced by PF6- were studied by the cyclic voltammetric methods.Experimental results indicated that the ion channel of BLM was open in the presence of the PF6- due to the interaction of PF6- with the BLM, while it was switched offin the absence of PF6-. Because the ion channel behavior was affected by the concentration of PF6-,a sensor for PF6- can be developed.

  7. Channels Formed by Botulinum, Tetanus, and Diphtheria Toxins in Planar Lipid Bilayers: Relevance to Translocation of Proteins across Membranes

    Science.gov (United States)

    Hoch, David H.; Romero-Mira, Miryam; Ehrlich, Barbara E.; Finkelstein, Alan; Dasgupta, Bibhuti R.; Simpson, Lance L.

    1985-03-01

    The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as ``tunnel proteins'' for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

  8. Point-Like Protrusion as a Prestalk Intermediate in Membrane Fusion Pathway

    OpenAIRE

    Efrat, Avishay; Chernomordik, Leonid V; Kozlov, Michael M.

    2007-01-01

    The widely accepted pathway of membrane fusion begins with the fusion stalk representing the initial intermediate of hemifusion. The lipid structures preceding hemifusion and their possible influence on fusion kinetics were not addressed. Here, we suggest the point-like protrusion as a prestalk fusion intermediate, which has energy lower than that of stalk and, therefore, does not limit the fusion rate. We demonstrate that by calculating the energy of the point-like protrusion, which depends ...

  9. Photodynamic inactivation of gramicidin channels in bilayer lipid membranes: protective efficacy of singlet oxygen quenchers depends on photosensitizer location.

    Science.gov (United States)

    Rokitskaya, T I; Firsov, A M; Kotova, E A; Antonenko, Y N

    2015-06-01

    The impact of double bonds in fatty acyl tails of unsaturated lipids on the photodynamic inactivation of ion channels formed by the pentadecapeptide gramicidin A in a planar bilayer lipid membrane was studied. The presence of unsaturated acyl tails protected gramicidin A against photodynamic inactivation, with efficacy depending on the depth of a photosensitizer in the membrane. The protective effect of double bonds was maximal with membrane-embedded chlorin e6-monoethylenediamine monoamide dimethyl ester, and minimal - in the case of water-soluble tri-sulfonated aluminum phthalocyanine (AlPcS3) known to reside at the membrane surface. By contrast, the protective effect of the hydrophilic singlet oxygen scavenger ascorbate was maximal for AlPcS3 and minimal for amide of chlorin e6 dimethyl ester. The depth of photosensitizer position in the lipid bilayer was estimated from the quenching of photosensitizer fluorescence by iodide. Thus, the protective effect of a singlet oxygen scavenger against photodynamic inactivation of the membrane-inserted peptide is enhanced upon location of the photosensitizer and scavenger molecules in close vicinity to each other. PMID:26531019

  10. Experimental Investigations of Direct and Converse Flexoelectric Effect in Bilayer Lipid Membranes.

    Science.gov (United States)

    Todorov, Angelio Todorov

    Flexoelectric coefficients (direct and converse), electric properties (capacitance and resistivity) and mechanical properties (thickness and elastic coefficients) have been determined for bilayer lipid membranes (BLMs) prepared from egg yolk lecithin (EYL), glycerol monoleate (GMO), phosphatidyl choline (PC) and phosphatidyl serine (PS) as a function of frequency, pH and surface charge modifiers. Direct flexoelectric effect manifested itself in the development of microvolt range a.c. potential (U_{f}) upon subjecting one side of a BLM to an oscillating hydrostatic pressure, in the 100-1000 Hz range. Operationally, the flexoelectric coefficient (f) is expressed by the ratio between U_{f} and the change of curvature (c) which accompanied the flexing of the membrane. Membrane curvature was determined by means of either the electric method (capacitance microphone effect) or by the newly developed method of stroboscopic interferometry. Real-time stroboscopic interferometry coupled with simultaneous electric measurements, provided a direct method for the determination of f. Two different frequency regimes of f were recognized. At low frequencies (GMO BLMs. At high frequencies (>300 Hz), associated with blocked mobility of the surfactant, f-values of 16.5 times 10^ {-19} and 0.30 times 10^{-19} Coulombs were obtained for PC and GMO BLMs. The theoretically calculated value for the GMO BLM oscillating at high frequency (0.12 times 10^{-19 } Coulombs) agreed well with that determined experimentally (0.3 times 10 ^{-19} Coulombs). For charged bovine brain PS BLM the observed flexocoefficient was f = 4.0 times 10^{ -18} Coulombs. Converse flexoelectric effect manifested itself in voltage-induced BLM curvature. Observations were carried out on uranyl acetate (UA) stabilized PS BLM under a.c. excitation. Frequency dependence of f was revealed by means of real-time stroboscopic interferometry. Satisfactory agreement was observed between the direct and converse f-values, measured

  11. Preparation and characterization of carbon molecular sieve (CMS) / SPEEK bilayer membranes and SPEEK / polyimide (PI) blend membranes for direct alcohols fuel cells / (DAFC) performance

    OpenAIRE

    Maab, Husnul

    2009-01-01

    Summary This research work is based on the preparation of membranes from SPEEK with reduced fuel cross-over following two different approach: 1) the preparation of bilayer membranes from in-home sulfonated PEEK (Vitrex) and a thin layer of in-home prepared carbon molecular sieve (CMS) from polyimide (Matrimid 5218); 2) blending of SPEEK with polyimide polymers. In both ways, it was possible to decrease the fuel cross-over in both direct methanol fuel cell (DMFC) and direct ethanol fuel ce...

  12. Study of pH (low) insertion peptides (pHLIPs) interaction with lipid bilayer of membrane

    Science.gov (United States)

    Weerakkody, Dhammika

    The pH-dependent interactions of pHLIPsRTM (pH (Low) Insertion Peptides) with lipid bilayer of membrane provides an opportunity to study and address fundamental questions of protein folding/insertion into membrane and unfolding/exit, as well as develop novel approach to target acidic diseased tissue such as cancer, ischemic myocardium, infection and others. The main goal of the work presented here is to answer the following questions: - What is the molecular mechanism of spontaneous insertion and folding of a peptide in a lipid bilayer of membrane; - What is the molecular mechanism of unfolding and exit of a peptide from a lipid bilayer of membrane; - How polar cargo attached to a peptide's inserting end might affect the process of insertion into a lipid bilayer of membrane; How sequence variation will affect a peptide's interactions with a lipid bilayer of membrane (partitioning into bilayer at neutral and low pH; apparent pK of insertion) with the main goal to identify the best pHLIP variants for imaging and therapy of pathological states such as cancer and others. It has been demonstrated that pHLIP insertion into a membrane is associated with the protonation of Asp/Glu residues, which leads to an increase of hydrophobicity that triggers the folding and insertion of the peptide across a lipid bilayer. The insertion of the pHLIP is unidirectional and it is accompanied by the release of energy. Therefore, the energy of membrane associated-folding can be used to favor the movement of cell-impermeable polar cargo molecules across the hydrophobic membrane bilayer when they are attached to the inserting end of pHLIP. Both pH-targeting behavior and molecular translocation have been demonstrated in cultured cells and in vivo. Thus, there is an opportunity to develop a novel concept in drug delivery, which is based on the use of a monomeric, pH-sensitive peptide molecular transporter, to deliver agents that are significantly more polar than conventional drugs

  13. A host–guest system to study structure–function relationships of membrane fusion peptides

    OpenAIRE

    Han, Xing; Tamm, Lukas K.

    2000-01-01

    We designed a host–guest fusion peptide system, which is completely soluble in water and has a high affinity for biological and lipid model membranes. The guest sequences are those of the fusion peptides of influenza hemagglutinin, which are solubilized by a highly charged unstructured C-terminal host sequence. These peptides partition to the surface of negatively charged liposomes or erythrocytes and elicit membrane fusion or hemolysis. They undergo a conformational ...

  14. Develop an artificial lipid bilayer array platform and screen nanoparticle-membrane interactions

    OpenAIRE

    Lu, Bin

    2014-01-01

    Artificial lipid bilayers have many uses. They are well established for scientific studies of reconstituted ion channels, used to host engineered pore proteins for sensing, especially DNA sequencing, and can potentially be applied in nanoparticle toxicity screening. To better explore these applications, we have simplified the formation and electrical measurement of the traditional method using an apparatus that only requires fluid dispensation. We achieved simultaneous bilayer formation and m...

  15. Protein secretion by hybrid bacterial ABC-transporters: specific functions of the membrane ATPase and the membrane fusion protein.

    OpenAIRE

    Binet, R; Wandersman, C

    1995-01-01

    The Erwinia chrysanthemi metalloprotease C and the Serratia marcescens haem acquisition protein HasA are both secreted from Gram-negative bacteria by a signal peptide-independent pathway which requires a C-terminal secretion signal and a specific ABC-transporter made up of three proteins: a membrane ATPase (the ABC-protein), a second inner membrane component belonging to the membrane fusion protein family and an outer membrane polypeptide. HasA and protease C transporters are homologous altho...

  16. The role of membrane viscosity in the dynamics of fluid membranes

    OpenAIRE

    Arroyo, Marino; DeSimone, Antonio; Heltai, Luca

    2010-01-01

    Fluid membranes made out of lipid bilayers are the fundamental separation structure in eukaryotic cells. Many physiological processes rely on dramatic shape and topological changes (e.g. fusion, fission) of fluid membrane systems. Fluidity is key to the versatility and constant reorganization of lipid bilayers. Here, we study the role of the membrane intrinsic viscosity, arising from the friction of the lipid molecules as they rearrange to accommodate shape changes, in the dynamics of morphol...

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

    Directory of Open Access Journals (Sweden)

    de la Vega Michelle

    2011-12-01

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

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

    Science.gov (United States)

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

    Science.gov (United States)

    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. PMID:26310672

  20. Scanning electrochemical microscopy. 38. Application of SECM to the study of charge transfer through bilayer lipid membranes.

    Science.gov (United States)

    Tsionsky, M; Zhou, J; Amemiya, S; Fan, F R; Bard, A J; Dryfe, R A

    1999-10-01

    The use of the scanning electrochemical microscope (SECM) to probe the kinetics of charge-transfer processes at bilayer lipid membranes (BLM) is presented. Analysis of the SECM tip response demonstrates that an unmodified BLM behaves as an insulator, whereas a BLM doped with iodine shows some positive feedback. The SECM technique thus allows one to probe processes at a BLM and determine the kinetics of the charge-transfer process. The SECM can also be used to determine the shape of the BLM. PMID:10660439

  1. Atomic Force Microscope Spectroscopy Reveals a Hemifusion Intermediate during Soluble N-Ethylmaleimide-Sensitive Factor-Attachment Protein Receptors-Mediated Membrane Fusion

    OpenAIRE

    Abdulreda, Midhat H.; Bhalla, Akhil; Chapman, Edwin R.; Moy, Vincent T.

    2007-01-01

    This study investigated the effect of soluble N-ethylmaleimide-sensitive factor-attachment protein (SNAP) receptors (SNAREs) on the fusion of egg L-α-phosphatidylcholine bilayers using atomic force microscope (AFM) spectroscopy. AFM measurements of the fusion force under compression were acquired to reveal the energy landscape of the fusion process. A single main energy barrier governing the fusion process was identified in the absence and presence of SNAREs in the bilayers. Under compression...

  2. Removal of the mechanoprotective influence of the cytoskeleton reveals PIEZO1 is gated by bilayer tension

    Science.gov (United States)

    Cox, Charles D.; Bae, Chilman; Ziegler, Lynn; Hartley, Silas; Nikolova-Krstevski, Vesna; Rohde, Paul R.; Ng, Chai-Ann; Sachs, Frederick; Gottlieb, Philip A.; Martinac, Boris

    2016-01-01

    Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensitive channel PIEZO1 is activated by force-transmission through the bilayer. To achieve this, we generate HEK293 cell membrane blebs largely free of cytoskeleton. Using the bacterial channel MscL, we calibrate the bilayer tension demonstrating that activation of MscL in blebs is identical to that in reconstituted bilayers. Utilizing a novel PIEZO1-GFP fusion, we then show PIEZO1 is activated by bilayer tension in bleb membranes, gating at lower pressures indicative of removal of the cortical cytoskeleton and the mechanoprotection it provides. Thus, PIEZO1 channels must sense force directly transmitted through the bilayer.

  3. Dynamic Heterogeneous Dielectric Generalized Born (DHDGB): An implicit membrane model with a dynamically varying bilayer thickness

    OpenAIRE

    Panahi, Afra; Feig, Michael

    2013-01-01

    An extension to the heterogeneous dielectric generalized Born (HDGB) implicit membrane formalism is presented to allow dynamic membrane deformations in response to membrane-inserted biomolecules during molecular dynamic simulations. The flexible membrane is implemented through additional degrees of freedom that represent the membrane deformation at the contact points of a membrane-inserted solute with the membrane. The extra degrees of freedom determine the dielectric and non-polar solvation ...

  4. A role for a TIMP-3-sensitive, Zn(2+)-dependent metalloprotease in mammalian gamete membrane fusion.

    Science.gov (United States)

    Correa, L M; Cho, C; Myles, D G; Primakoff, P

    2000-09-01

    During fertilization, sperm and egg plasma membranes adhere and then fuse by a mechanism that is not well understood. Zinc metalloproteases are necessary for some intercellular fusion events, for instance, cell-cell fusion in yeast. In this study we tested the effects of class-specific and family-specific protease inhibitors on mouse gamete fusion. Capacitated, acrosome-reacted sperm and zona-free eggs were used in assays designed to define the effects of inhibitors on sperm-egg plasma membrane binding or fusion. Inhibitors of the aspartic, cysteine, and serine protease classes had no effect on sperm-egg binding or fusion. Both a synthetic metalloprotease substrate (succinyl-Ala-Ala-Phe-amidomethylcoumarin) and the zinc chelator 1,10-phenanthroline inhibited sperm-egg fusion but did not decrease sperm-egg binding. The fusion-inhibition effect of phenanthroline was reversible and activity of the inhibitable zinc metalloprotease was shown to be required during a short time window, the first 15 min after insemination. Tissue inhibitor of metalloprotease-3 and Ro 31-9790, specific inhibitors of zinc metalloproteases in the matrixin and adamalysin families, also inhibited sperm-egg fusion but not sperm-egg binding. These data indicate a role in gamete fusion for one or more zinc metalloproteases of the matrixin and/or adamalysin families that act after plasma membrane binding and before sperm-egg membrane fusion. PMID:10964469

  5. [The effect of the homogenates from different developmental stages of the nematode Protostrongylus rufescens (Leuckart, 1895) on mitochondrial and lipid bilayer membranes].

    Science.gov (United States)

    Kuchboev, A E; Kazakov, I; Asrarov, M I; Isakova, D T; Azimov, D A; Golovanov, V I

    2007-01-01

    The effect of the homogenates from different developmental stages of the nematode Protostrongylus rufescens on mitochondrial and lipid bilayer membranes has been studied. The homogenate of P. rufescens affects efficiently the cell energy by the inhibition of the mitochondrial respiration in the metabolic state V3, uncouples oxidative phosphorylation and affects the functions of mitochondria at the level of cyclosporine A-sensitive pore by making it highly permeable. Moreover, the nematode homogenate at the concentration of 1 mkg/ml increases efficiently the integral permeability of lipid bilayer membranes. An increase in this permeability is connected apparently with the formation of single ion channels. The channels of lipid bilayer membranes induced by the nematode homogenate show cation selectivity. PMID:17460939

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

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-01-01

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

  7. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

    Energy Technology Data Exchange (ETDEWEB)

    Ding, Yi; Fujimoto, L. Miya; Yao, Yong; Marassi, Francesca M., E-mail: fmarassi@sbmri.org [Sanford-Burnham Medical Research Institute (United States)

    2015-04-15

    Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales.

  8. Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation

    International Nuclear Information System (INIS)

    Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396–10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure–activity correlation experiments across a wide range of timescales

  9. A possible role of rhodopsin in maintaining bilayer structure in the photoreceptor membrane

    NARCIS (Netherlands)

    Grip, W.J. de; Drenthe, E.H.S.; Echteld, C.J.A. van; Kruijff, B. de; Verkleij, A.J.

    1979-01-01

    31P-NMR measurements demonstrate that at 37°C, independent of the photolytic state of the photopigment rhodopsin, the lipids in the photoreceptormembrane are almost exclusively organised in a bilayer. In strong contrast, the 31P-NMR spectra of the extracted lipids are characteristic for the hexagona

  10. Supported lipid bilayer on nanocrystalline diamond: dual optical and field-effect sensor for membrane disruption

    Czech Academy of Sciences Publication Activity Database

    Ang, P.K.; Loh, K.P.; Wohland, T.; Nesládek, Miloš; Van Hove, E.

    2009-01-01

    Roč. 19, č. 1 (2009), s. 109-116. ISSN 1616-301X Institutional research plan: CEZ:AV0Z10100520 Keywords : nanocrystalline diamond * biocompatibility * supported lipid bilayers * biosensors * solution gate field effect transistor Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 6.990, year: 2009

  11. Bilayer vesicles of amphiphilic cyclodextrines: host membranes that recognize guest molecules

    NARCIS (Netherlands)

    Falvey, P.; Lim, C.W.; Darcy, R.; Revermann, T.; Karst, U.; Marcelis, A.T.M.; Coleman, A.W.; Reinhoudt, D.N.; Ravoo, B.J.

    2005-01-01

    A family of amphiphilic cyclodextrins (6, 7) has been prepared through 6-S-alkylation (alkyl=n-dodecyl and n-hexadecyl) of the primary side and 2-O-PEGylation of the secondary side of alpha-, beta-, and gamma-cyclodextrins (PEG=poly(ethylene glycol)). These cyclodextrins form nonionic bilayer vesicl

  12. Bilayer Vesicles of Amphiphilic Cyclodextrins: Host Membranes That Recognize Guest Molecules

    NARCIS (Netherlands)

    Falvey, Patrick; Lim, Choon Woo; Darcy, Raphael; Revermann, Tobias; Karst, Uwe; Giesbers, Marcel; Marcelis, Antonius T.M.; Lazar, Adina; Coleman, Anthony W.; Reinhoudt, David N.; Ravoo, Bart Jan

    2005-01-01

    A family of amphiphilic cyclodextrins (6, 7) has been prepared through 6-S-alkylation (alkyl=n-dodecyl and n-hexadecyl) of the primary side and 2-O-PEGylation of the secondary side of a-, B-, and Y-cyclodextrins (PEG=poly(ethylene glycol)). These cyclodextrins form nonionic bilayer vesicles in aqueo

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

    Directory of Open Access Journals (Sweden)

    Cécile Sauvanet

    Full Text Available 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

  14. The influence of oscillating electromagnetic fields on membrane structure and function: Synthetic liposome and natural membrane bilayer systems with direct application to the controlled delivery of chemical agents

    International Nuclear Information System (INIS)

    Investigations have been conducted to determine if an imposed electromagnetic field can influence membrane transport, and ion and drug permeability in both synthetic and natural cell membrane systems. Microwave fields enhance accumulation of sodium in the lymphocyte and induce protein shedding at Tc. Microwaves also trigger membrane permeability of liposome systems under specific field exposure conditions. Sensitivity varies in a defined way in bilayers displaying a membrane structural phase transition temperature, Tc; maximal release was observed at or near Tc. Significantly, liposome systems without a membrane phase transition were also found to experience permeability increases but, in contrast, this response was temperature independent. The above results indicate that field-enhanced drug release occurs in liposome vesicles that possess a Tc as well as non-Tc liposomes. Additional studies extend non-Tc liposome responses to the in vivo case in which microwaves trigger Gentamicin release from a liposome ''depot'' placed subcutaneously in the rat hind leg. In addition, evidence is provided that cell surface sequestered liposomes can be triggered by microwave fields to release drugs directly into target cells. 24 refs., 6 figs

  15. All electrochemical fabrication of a bilayer membrane composed of nanotubular photocatalyst and palladium toward high-purity hydrogen production

    Science.gov (United States)

    Hattori, Masashi; Noda, Kei

    2015-12-01

    We developed an all-electrochemical technique for fabricating a bilayer structure of a titanium dioxide (TiO2) nanotube array (TNA) and a palladium film (TNA/Pd membrane), which works for photocatalytic high-purity hydrogen production. Electroless plating was used for depositing the Pd film on the TNA surface prepared by anodizing a titanium foil. A 3-μm-thick TNA/Pd membrane without any pinholes in a 1.5-cm-diameter area was fabricated by transferring a 1-μm-thick TNA onto an electroless-plated 2-μm-thick Pd film with a mechanical peel-off process. This ultrathin membrane with sufficient mechanical robustness showed photocatalytic H2 production via methanol reforming under ultraviolet illumination on the TNA side, immediately followed by the purification of the generated H2 gas through the Pd layer. The hydrogen production rate and the apparent quantum yield for high-purity H2 production from methanol/water mixture with the TNA/Pd membrane were also examined. This work suggests that palladium electroless plating is more suitable and practical for preparing a well-organized TNA/Pd heterointerface than palladium sputter deposition.

  16. Formaldehyde assay by capacitance versus voltage and impedance measurements using bi-layer bio-recognition membrane.

    Science.gov (United States)

    Ben Ali, M; Korpan, Y; Gonchar, M; El'skaya, A; Maaref, M A; Jaffrezic-Renault, N; Martelet, C

    2006-12-15

    A novel formaldehyde sensitive biosensor based on bacterial formaldehyde dehydrogenase (FDH) as a bio-recognition element has been developed. The bio-recognition membrane had bi-layer architecture and consisted of FDH, cross-linked with albumin, and of the cofactor NAD at a high concentration level (first layer). The second layer was a negatively charged Nafion membrane, which prevented a leakage of negatively charged NAD molecules from the bio-membrane. As transducers, gold electrodes SiO(2)/Si/SiO(2)/Ti/Au and electrolyte-insulator-semiconductor Si/SiO(2) (EIS) structures have been used. Changes in capacitance and impedance properties of the bio-recognition membrane have been used for monitoring formaldehyde concentration in a bulk solution. It has been shown that formaldehyde can be detected within a concentration range from 1 microM to 20mM depending on the type of transduction used, with a detection limit of 1 and 100 microM for gold-based and EIS-based transducers, respectively. PMID:16516460

  17. The quantum casimir effect may be a universal force organizing the bilayer structure of the cell membrane.

    Science.gov (United States)

    Pawlowski, Piotr H; Zielenkiewicz, Piotr

    2013-05-01

    A mathematic-physical model of the interaction between cell membrane bilayer leaflets is proposed based on the Casimir effect in dielectrics. This model explains why the layers of a lipid membrane gently slide one past another rather than penetrate each other. The presented model reveals the dependence of variations in the free energy of the system on the membrane thickness. This function is characterized by the two close minima corresponding to the different levels of interdigitation of the lipids from neighbor layers. The energy barrier of the compressing transition between the predicted minima is estimated to be 5.7 kT/lipid, and the return energy is estimated to be 3.1 kT/lipid. The proposed model enables estimation of the value of the membrane elastic thickness modulus of compressibility, which is 1.7 × 10⁹ N/m², and the value of the interlayer friction coefficient, which is 1.9 × 10⁸ Ns/m³. PMID:23612889

  18. On the Fusion of Confucianism, Buddhism and Taoism in Chinese Cognitive Membrane

    OpenAIRE

    Jiawei Geng; H. J. Cai

    2014-01-01

    The coexistence of Confucianism, Buddhism and Taoism in China is phenomenal through world cultural history, which was explained by putting emphasis on the particularity or complementarity of those three doctrines. We proposed that, Chinese long-term self-assertiveness demands and their evolution lead to enduring competitions among them and eventual fusion of them, within Chinese Cognitive Membrane. It is emphasized that Chinese Cognitive Membrane needs further fusion with spirits of modern sc...

  19. Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

    OpenAIRE

    Kosower, N S; Glaser, T; Kosower, E M

    1983-01-01

    Rat, but not human, erythrocytes undergo fusion promoted by the membrane-mobility agent 2-(2-methoxyethoxy)-ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C). The difference in behavior is correlated with rat erythrocyte membrane protein degradation caused by Ca2+-activated proteases. The human erythrocyte is deficient in such protease activity. Membrane protein degradation is a necessary, but not sufficient, requirement for membrane fusion. Membrane protein degradation probably releases membra...

  20. Membrane pumping technology for helium and hydrogen isotope separation in the fusion reactor

    Energy Technology Data Exchange (ETDEWEB)

    Pistunovich, V.I. [Kurchatov Inst., Moscow (Russian Federation). NFI RRC; Pigarov, A.Yu. [Kurchatov Inst., Moscow (Russian Federation). NFI RRC; Busnyuk, A.O. [Bonch-Bruyevich University, St. Petersburg (Russian Federation); Livshits, A.I. [Bonch-Bruyevich University, St. Petersburg (Russian Federation); Notkin, M.E. [Bonch-Bruyevich University, St. Petersburg (Russian Federation); Samartsev, A.A. [Bonch-Bruyevich University, St. Petersburg (Russian Federation); Borisenko, K.L. [Efremov Institute, St. Petersburg (Russian Federation); Darmogray, V.V. [Efremov Institute, St. Petersburg (Russian Federation); Ershov, B.D. [Efremov Institute, St. Petersburg (Russian Federation); Filippova, L.V. [Efremov Institute, St. Petersburg (Russian Federation); Mudugin, B.G. [Efremov Institute, St. Petersburg (Russian Federation); Odintsov, V.N. [Efremov Institute, St. Petersburg (Russian Federation); Saksagansky, G.L. [Efremov Institute, St. Petersburg (Russian Federation); Serebrennikov, D.V. [Efremov Institute, St. Petersburg (Russian Federation)

    1995-03-01

    A gas pumping system for ITER, improved by implementation of superpermeable membranes for selective hydrogen isotope exhaust, is considered. A study of the pumping capability of a niobium membrane for a hydrogen-helium mixture has been performed.Monte Carlo simulations of gas behaviour for the experimental facility and fusion reactor have been done.The scheme of the ITER pumping system with the membranes and membrane pumping technology was considered. The conceptual study the membrane pump for the ITER was done. This work gives good prospects for the membrane pumping use in ITER to reduce the total inventory of tritium necessary for reactor operation. (orig.).

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

    Energy Technology Data Exchange (ETDEWEB)

    Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

    2013-04-05

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

  2. Cell-based analysis of Chikungunya virus E1 protein in membrane fusion

    Directory of Open Access Journals (Sweden)

    Kuo Szu-Cheng

    2012-04-01

    Full Text Available Abstract Background Chikungunya fever is a pandemic disease caused by the mosquito-borne Chikungunya virus (CHIKV. E1 glycoprotein mediation of viral membrane fusion during CHIKV infection is a crucial step in the release of viral genome into the host cytoplasm for replication. How the E1 structure determines membrane fusion and whether other CHIKV structural proteins participate in E1 fusion activity remain largely unexplored. Methods A bicistronic baculovirus expression system to produce recombinant baculoviruses for cell-based assay was used. Sf21 insect cells infected by recombinant baculoviruses bearing wild type or single-amino-acid substitution of CHIKV E1 and EGFP (enhanced green fluorescence protein were employed to investigate the roles of four E1 amino acid residues (G91, V178, A226, and H230 in membrane fusion activity. Results Western blot analysis revealed that the E1 expression level and surface features in wild type and mutant substituted cells were similar. However, cell fusion assay found that those cells infected by CHIKV E1-H230A mutant baculovirus showed little fusion activity, and those bearing CHIKV E1-G91D mutant completely lost the ability to induce cell-cell fusion. Cells infected by recombinant baculoviruses of CHIKV E1-A226V and E1-V178A mutants exhibited the same membrane fusion capability as wild type. Although the E1 expression level of cells bearing monomeric-E1-based constructs (expressing E1 only was greater than that of cells bearing 26S-based constructs (expressing all structural proteins, the sizes of syncytial cells induced by infection of baculoviruses containing 26S-based constructs were larger than those from infections having monomeric-E1 constructs, suggesting that other viral structure proteins participate or regulate E1 fusion activity. Furthermore, membrane fusion in cells infected by baculovirus bearing the A226V mutation constructs exhibited increased cholesterol-dependences and lower pH thresholds

  3. Biological Evaluation (In Vitro and In Vivo) of Bilayered Collagenous Coated (Nano Electrospun and Solid Wall) Chitosan Membrane for Periodontal Guided Bone Regeneration.

    Science.gov (United States)

    Lotfi, Ghogha; Shokrgozar, Mohammad Ali; Mofid, Rasoul; Abbas, Fatemeh Mashhadi; Ghanavati, Farzin; Baghban, Alireza Akbarzadeh; Yavari, Seyedeh Kimia; Pajoumshariati, Seyedramin

    2016-07-01

    The application of barrier membranes in guided bone regeneration (GBR) has become a commonly used surgical technique in periodontal research. The objectives of this study were to evaluate the in vitro biocompatibility and osteogenic differentiation of mesenchymal stem cells (MSCs) on two different collagenous coatings (nano electrospun fibrous vs. solid wall) of bilayered collagen/chitosan membrane and their histological evaluation on bone regeneration in rabbit calvarial defects. It was found that chitosan-nano electrospun collagen (CNC) membranes had higher proliferation/metabolic activity compared to the chitosan-collagen (CC) and pristine chitosan membranes. The qRT-PCR analysis demonstrated the CNC membranes induced significant expression of osteogenic genes (Osteocalcin, RUNX2 and Col-α1) in MSCs. Moreover, higher calcium content and alkaline phosphatase activity of MSCs were observed compared to the other groups. Histologic and histomorphometric evaluations were performed on the uncovered (negative control) as well as covered calvarial defects of ten adult white rabbits with different membranes (CNC, CC, BioGide (BG, positive control)) at 1 and 2 months after surgery. More bone formation was detected in the defects covered with CNC and BG membranes than those covered by CC and the negative control. No inflammation and residual biomaterial particles were observed on the membrane surface or in the surrounding tissues in the surgical areas. These results suggest that bilayer CNC membrane can have the potential for use as a GBR membrane material facilitating bone formation. PMID:26586588

  4. Geometry of the Contact Zone between Fused Membrane-Coated Beads Mimicking Cell-Cell Fusion.

    Science.gov (United States)

    Savić, Filip; Kliesch, Torben-Tobias; Verbeek, Sarah; Bao, Chunxiao; Thiart, Jan; Kros, Alexander; Geil, Burkhard; Janshoff, Andreas

    2016-05-24

    The fusion of lipid membranes is a key process in biology. It enables cells and organelles to exchange molecules with their surroundings, which otherwise could not cross the membrane barrier. To study such complex processes we use simplified artificial model systems, i.e., an optical fusion assay based on membrane-coated glass spheres. We present a technique to analyze membrane-membrane interactions in a large ensemble of particles. Detailed information on the geometry of the fusion stalk of fully fused membranes is obtained by studying the diffusional lipid dynamics with fluorescence recovery after photobleaching experiments. A small contact zone is a strong obstruction for the particle exchange across the fusion spot. With the aid of computer simulations, fluorescence-recovery-after-photobleaching recovery times of both fused and single-membrane-coated beads allow us to estimate the size of the contact zones between two membrane-coated beads. Minimizing delamination and bending energy leads to minimal angles close to those geometrically allowed. PMID:27224487

  5. Construction of a patterned hydrogel-fibrous mat bilayer structure to mimic choroid and Bruch's membrane layers of retina.

    Science.gov (United States)

    Komez, Aylin; Baran, Erkan T; Erdem, Uzeyir; Hasirci, Nesrin; Hasirci, Vasif

    2016-09-01

    Deterioration of retina and death of the retinal cells due to age, diabetes, or occlusion can cause retinal degeneration which leads to loss of vision. In this study, it is aimed to design a bilayered matrix to mimic the choroid and the Bruch's membrane of the retinal tissue. As choroid, a microchanneled network resembling a fractal tree design was fabricated by photolithography over photo-cross-linkable methacrylated hyaluronic acid hydrogel. Gelatin or collagen was immobilized into the microchannels to enhance adherence of Human Umbilical Vein Endothelial Cells (HUVEC). At late culture periods (2 weeks), formation of tubular structures due to proliferation of the attached cells was observed. As Bruch's membrane, an electrospun fibroin nanofiber mat was produced to grow retinal pigment epithelium (RPE) cells on. Cellular interactions between RPE and HUVEC in the microchannels were investigated in a coculture model in a noncontact mode. It was deduced that by combining the RPE layer on the highly permeable Bruch's membrane with the choroid layer populated by HUVECs, a retinal substitute which might have a potential for use in the treatment of retinal diseases is possible. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2166-2177, 2016. PMID:27102366

  6. (13) C-TmDOTA as versatile thermometer compound for solid-state NMR of hydrated lipid bilayer membranes.

    Science.gov (United States)

    Umegawa, Yuichi; Tanaka, Yuya; Nobuaki, Matsumori; Murata, Michio

    2016-03-01

    Recent advances in solid-state nuclear magnetic resonance (NMR) techniques, such as magic angle spinning and high-power decoupling, have dramatically increased the sensitivity and resolution of NMR. However, these NMR techniques generate extra heat, causing a temperature difference between the sample in the rotor and the variable temperature gas. This extra heating is a particularly crucial problem for hydrated lipid membrane samples. Thus, to develop an NMR thermometer that is suitable for hydrated lipid samples, thulium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (TmDOTA) was synthesized and labeled with (13) C (i.e., (13) C-TmDOTA) to increase the NMR sensitivity. The complex was mixed with a hydrated lipid membrane, and the system was subjected to solid-state NMR and differential scanning calorimetric analyses. The physical properties of the lipid bilayer and the quality of the NMR spectra of the membrane were negligibly affected by the presence of (13) C-TmDOTA, and the (13) C chemical shift of the complex exhibited a large-temperature dependence. The results demonstrated that (13) C-TmDOTA could be successfully used as a thermometer to accurately monitor temperature changes induced by (1) H decoupling pulses and/or by magic angle spinning and the temperature distribution of the sample inside the rotor. Thus, (13) C-TmDOTA was shown to be a versatile thermometer for hydrated lipid assemblies. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26460094

  7. Marine sponge cyclic peptide theonellamide A disrupts lipid bilayer integrity without forming distinct membrane pores.

    Science.gov (United States)

    Espiritu, Rafael Atillo; Cornelio, Kimberly; Kinoshita, Masanao; Matsumori, Nobuaki; Murata, Michio; Nishimura, Shinichi; Kakeya, Hideaki; Yoshida, Minoru; Matsunaga, Shigeki

    2016-06-01

    Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides derived from the marine sponge Theonella sp. These peptides specifically bind to 3β-hydroxysterols, resulting in 1,3-β-d-glucan overproduction and membrane damage in yeasts. The inclusion of cholesterol or ergosterol in phosphatidylcholine membranes significantly enhanced the membrane affinity of theonellamide A (TNM-A) because of its direct interaction with 3β-hydroxyl groups of sterols. To better understand TNM-induced membrane alterations, we investigated the effects of TNM-A on liposome morphology. (31)P nuclear magnetic resonance (NMR) and dynamic light scattering (DLS) measurements revealed that the premixing of TNM-A with lipids induced smaller vesicle formation. When giant unilamellar vesicles were incubated with exogenously added TNM-A, confocal micrographs showed dynamic changes in membrane morphology, which were more frequently observed in cholesterol-containing than sterol-free liposomes. In conjunction with our previous data, these results suggest that the membrane action of TNM-A proceeds in two steps: 1) TNM-A binds to the membrane surface through direct interaction with sterols and 2) accumulated TNM-A modifies the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption. PMID:27003125

  8. Microfabricated Teflon Membranes for Low-Noise Recordings of Ion Channels in Planar Lipid Bilayers

    OpenAIRE

    Mayer, Michael; Kriebel, Jennah K.; Tosteson, Magdalena T.; Whitesides, George M.

    2003-01-01

    We present a straightforward, accessible method for the fabrication of micropores with diameters from 2 to 800 μm in films of amorphous Teflon (Teflon AF). Pores with diameters ≤40 μm made it possible to record ion fluxes through ion channels in planar bilayers with excellent signal characteristics. These pores afforded: i), stable measurements at transmembrane voltages up to 460 mV; ii), recordings at low noise levels (0.4 pA rms at 4.3 kHz bandwidth); iii), recordings at high effective band...

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

    OpenAIRE

    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-c...

  10. Stress analysis and fail-safe design of bilayered tubular supported ceramic membranes

    DEFF Research Database (Denmark)

    Kwok, Kawai; Frandsen, Henrik Lund; Søgaard, Martin; Hendriksen, Peter Vang

    2014-01-01

    Supported ceramic membranes based on mixed ionic and electronic conductors are a promising technology for oxygen separation applications. In addition to chemically induced stress under oxygen activity gradients in the materials, strain mismatch between membrane and support gives rise to considera......Supported ceramic membranes based on mixed ionic and electronic conductors are a promising technology for oxygen separation applications. In addition to chemically induced stress under oxygen activity gradients in the materials, strain mismatch between membrane and support gives rise to....... Stress distributions in two membrane systems have been analyzed and routes to minimize stress are proposed. For a Ba0.5Sr0.5Co0.8Fe0.2O3−δBa0.5Sr0.5Co0.8Fe0.2O3−δ membrane supported on a porous substrate of the same material under pressure-vacuum operation, the optimal configuration in terms of...

  11. Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

    OpenAIRE

    Ramakrishnan, N.; Sunil Kumar, P. B.; Radhakrishnan, Ravi

    2014-01-01

    Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to ...

  12. Formation, Stability, and Mobility of One-Dimensional Lipid Bilayer on High Curvature Substrates

    Energy Technology Data Exchange (ETDEWEB)

    Huang, J; Martinez, J; Artyukhin, A; Sirbuly, D; Wang, Y; Ju, J W; Stroeve, P; Noy, A

    2007-03-23

    Curved lipid membranes are ubiquitous in living systems and play an important role in many biological processes. To understand how curvature and lipid composition affect membrane formation and fluidity we have assembled and studied mixed 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) and 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE) supported lipid bilayers on amorphous silicon nanowires with controlled diameters ranging from 20 nm to 200 nm. Addition of cone-shaped DOPE molecules to cylindrical DOPC molecules promotes vesicle fusion and bilayer formation on smaller diameter nanowires. Our experiments demonstrate that nanowire-supported bilayers are mobile, exhibit fast recovery after photobleaching, and have low concentration of defects. Lipid diffusion coefficients in these high-curvature tubular membranes are comparable to the values reported for flat supported bilayers and increase with decreasing nanowire diameter.

  13. Spatial distribution and activity of Na(+)/K(+)-ATPase in lipid bilayer membranes with phase boundaries.

    Science.gov (United States)

    Bhatia, Tripta; Cornelius, Flemming; Brewer, Jonathan; Bagatolli, Luis A; Simonsen, Adam C; Ipsen, John H; Mouritsen, Ole G

    2016-06-01

    We have reconstituted functional Na(+)/K(+)-ATPase (NKA) into giant unilamellar vesicles (GUVs) of well-defined binary and ternary lipid composition including cholesterol. The activity of the membrane system can be turned on and off by ATP. The hydrolytic activity of NKA is found to depend on membrane phase, and the water relaxation in the membrane on the presence of NKA. By collapsing and fixating the GUVs onto a solid support and using high-resolution atomic-force microscopy (AFM) imaging we determine the protein orientation and spatial distribution at the single-molecule level and find that NKA is preferentially located at lo/ld interfaces in two-phase GUVs and homogeneously distributed in single-phase GUVs. When turned active, the membrane is found to unbind from the support suggesting that the protein function leads to softening of the membrane. PMID:26994932

  14. Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion

    International Nuclear Information System (INIS)

    Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a 'pore'-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell

  15. Cationic Au Nanoparticle Binding with Plasma Membrane-like Lipid Bilayers: Potential Mechanism for Spontaneous Permeation to Cells Revealed by Atomistic Simulations

    DEFF Research Database (Denmark)

    Heikkila, E.; Martinez-Seara, H.; Gurtovenko, A. A.; Javanainen, M.; Hakkinen, H.; Vattulainen, I.; Akola, J.

    2014-01-01

    Au nanoparticles interacting with realistic membranes and explicit solvent using a model system that comprises two cellular compartments, extracellular and cytosolic, divided by two asymmetric lipid bilayers. The membrane-AuNP+ binding and membrane reorganization processes are discovered to be...... governed by cooperative effects where AuNP+, counterions, water, and the two membrane leaflets all contribute. On the extracellular side, we find that the nanoparticle has to cross a free energy barrier of about 5 k(B)T prior forming a stable contact with the membrane. This results in a rearrangement of...... the zwitterionic lipids and nanoparticle side groups in the contact area, giving rise to the initial stage of pore formation on the membrane surface. Such behavior is not seen on the cytosolic side, where AuNP+ is spontaneously captured by the negatively charged phosphatidylserine lipids that diffuse...

  16. Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

    CERN Document Server

    Ramakrishnan, N; Radhakrishnan, Ravi

    2015-01-01

    Biological membranes constitute boundaries of cells and cell organelles. Physico-chemical mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. The suite of methods discussed here can be tailored to applicatio...

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

    Directory of Open Access Journals (Sweden)

    Bin Lu

    2015-06-01

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

  18. Quantitative optical microscopy and micromanipulation studies on the lipid bilayer membranes of giant unilamellar vesicles

    DEFF Research Database (Denmark)

    Bagatolli, Luis; Needham, David

    2014-01-01

    This manuscript discusses basic methodological aspects of optical microscopy and micromanipulation methods to study membranes and reviews methods to generate giant unilamellar vesicles (GUVs). In particular, we focus on the use of fluorescence microscopy and micropipette manipulation techniques to...... experimentation. For instance, using different fluorescent reporters, fluorescence microscopy allows strategies to study membrane lateral structure/dynamics at the level of single vesicles of diverse compositions. The micropipette manipulation technique on the other hand, uses Hoffman modulation contrast...

  19. Engineering hybrid exosomes by membrane fusion with liposomes

    OpenAIRE

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

    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 modifi...

  20. Size-selective permeation of water-soluble polymers through the bilayer membrane of cyclodextrin vesicles investigated by PFG-NMR.

    Science.gov (United States)

    Himmelein, Sabine; Sporenberg, Nora; Schönhoff, Monika; Ravoo, Bart Jan

    2014-04-15

    Cyclodextrin vesicles (CDVs) consist of a bilayer of amphiphilic cyclodextrins (CDs). CDVs exhibit CD cavities at their surface that are able to recognize and bind hydrophobic guest molecules via size-selective inclusion. In this study, the permeability of α- and β-CDVs is investigated by pulsed field gradient-stimulated echo (PFG-STE) nuclear magnetic resonance. Diffusion experiments with water and two types of water-soluble polymers, polyethylene glycol (PEG) and polypropylene glycol (PPG), revealed three main factors that influence the exchange rate and permeability of CDVs. First, the length of the hydrophobic chain of the CD amphiphile plays a crucial role. Reasonably, vesicles consisting of amphiphiles with a longer aliphatic chain are less permeable since both membrane thickness and melting temperature T(m) increase. Second, the exchange rate through the bilayer membrane depends on the molecular weight of the polymer and decreases with increasing weight of the polymer. Most interestingly, a size-selective distinction of permeation due to the embedded CDs in the bilayer membrane was found. The mechanism of permeation is shown to occur through the CD cavity, such that depending on the size of the cavity, permeation of polymers with different cross-sectional diameters takes place. Whereas PPG permeates through the membrane of β-CD vesicles, it does not permeate α-CD vesicles. PMID:24650278

  1. Effect of antimicrobial peptide on the dynamics of phosphocholine membrane: role of cholesterol and physical state of bilayer.

    Science.gov (United States)

    Sharma, V K; Mamontov, E; Anunciado, D B; O'Neill, H; Urban, V S

    2015-09-14

    Antimicrobial peptides are universal in all forms of life and are well known for their strong interaction with the cell membrane. This makes them a popular target for investigation of peptide-lipid interactions. Here we report the effect of melittin, an important antimicrobial peptide, on the dynamics of membranes based on 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid in both the solid gel and fluid phases. To probe the phase transition, elastic neutron intensity temperature scans have been carried out on DMPC-based unilamellar vesicles (ULV) with and without melittin. We have found that addition of a small amount (0.2 mol%) melittin eliminates the steep fall in the elastic intensity at 296 K associated with the solid gel to fluid phase transition, which is observed for pure DMPC vesicles. Quasielastic neutron scattering (QENS) experiments have been carried out on DMPC ULV in the solid gel and fluid phases with and without 0.2 mol% melittin. The data analysis invariably shows the presence of lateral and internal motions of the DMPC molecule. We found that melittin does have a profound effect on the dynamics of lipid molecules, especially on the lateral motion, and affects it in a different way, depending on the phase of the bilayers. In the solid gel phase, it acts as a plasticizer, enhancing the lateral motion of DMPC. However, in the fluid phase it acts as a stiffening agent, restricting the lateral motion of the lipid molecules. These observations are consistent with the mean squared displacements extracted from the elastic intensity temperature scans. Their importance lies in the fact that many membrane processes, including signaling and energy transduction pathways, are controlled to a great extent by the lateral diffusion of lipids in the membrane. To investigate the effect of melittin on vesicles supplemented with cholesterol, QENS experiments have also been carried out on DMPC ULV with cholesterol in the presence and absence of 0.2 mol% melittin

  2. On the dynamics of molecular self-assembly and the structural analysis of bilayer membranes using coarse-grained molecular dynamics simulations.

    Science.gov (United States)

    Schindler, Tanja; Kröner, Dietmar; Steinhauser, Martin O

    2016-09-01

    We present a molecular dynamics simulation study of the self-assembly of coarse-grained lipid molecules from unbiased random initial configurations. Our lipid model is based on a well-tried CG polymer model with an additional potential that mimics the hydrophobic properties of lipid tails. We find that several stages of self-organization of lipid clusters are involved in the dynamics of bilayer formation and that the resulting equilibrium structures sensitively depend on the strength of hydrophobic interactions hc of the lipid tails and on temperature T. The obtained stable lipid membranes are quantitatively analyzed with respect to their local structure and their degree of order. At equilibrium, we obtain self-stabilizing bilayer membrane structures that exhibit a bending stiffness κB and compression modulus KC comparable to experimental measurements under physiological conditions. We present a phase diagram of our lipid model which covers a sol-gel transition, a liquid (or gel-like) phase including stable bilayer structures and vesicle formation, as well as a quasi-crystalline phase. We also determine the exact conditions for temperature T and degree of hydrophobicity hc for stable bilayer formation including closed vesicles. PMID:27216316

  3. The fusion-related hydrophobic domain of Sendai F protein can be moved through the cytoplasmic membrane of Escherichia coli.

    OpenAIRE

    Davis, N G; Hsu, M. C.

    1986-01-01

    Recent work on a prokaryotic membrane protein, gene III protein (pIII) of coliphage f1, showed that polypeptide segments of sufficient hydrophobicity functioned to stop transfer of the polypeptide across the cell membrane: strings of 16 or more hydrophobic amino acids sufficed. A fusion-related hydrophobic domain (FRHD) of Sendai F protein, a sequence of 26 consecutive uncharged residues, has been implicated in the fusion of the viral membrane envelope and the target-cell membrane through a h...

  4. GPI-anchored influenza hemagglutinin induces hemifusion to both red blood cell and planar bilayer membranes

    OpenAIRE

    1995-01-01

    Under fusogenic conditions, fluorescent dye redistributed from the outer monolayer leaflet of red blood cells (RBCs) to cells expressing glycophosphatidylinositol-anchored influenza virus hemagglutinin (GPI- HA) without transfer of aqueous dye. This suggests that hemifusion, but not full fusion, occurred (Kemble, G. W., T. Danieli, and J. M. White. 1994. Cell. 76:383-391). We extended the evidence for hemifusion by labeling the inner monolayer leaflets of RBCs with FM4-64 and observing that t...

  5. Fusion-Triggered Switching of Enzymatic Activity on an Artificial Cell Membrane

    Directory of Open Access Journals (Sweden)

    Jun-ichi Kikuchi

    2012-05-01

    Full Text Available A nanosensory membrane device was constructed for detecting liposome fusion through changes in an enzymatic activity. Inspired by a biological signal transduction system, the device design involved functionalized liposomal membranes prepared by self-assembly of the following molecular components: a synthetic peptide lipid and a phospholipid as matrix membrane components, a Schiff’s base of pyridoxal 5’-phosphate with phosphatidylethanolamine as a thermo-responsive artificial receptor, NADH-dependent L-lactate dehydrogenase as a signal amplifier, and Cu2+ ion as a signal mediator between the receptor and enzyme. The enzymatic activity of the membrane device was adjustable by changing the matrix lipid composition, reflecting the thermotropic phase transition behavior of the lipid membranes, which in turn controlled receptor binding affinity toward the enzyme-inhibiting mediator species. When an effective fusogen anionic polymer was added to these cationic liposomes, membrane fusion occurred, and the functionalized liposomal membranes responded with changes in enzymatic activity, thus serving as an effective nanosensory device for liposome fusion detection.

  6. Solution NMR of membrane proteins in bilayer mimics: Small is beautiful, but sometimes bigger is better

    Science.gov (United States)

    Poget, Sébastien F.; Girvin, Mark E.

    2007-01-01

    Considerable progress has been made recently on solution NMR studies of multi-transmembrane helix membrane protein systems of increasing size. Careful correlation of structure with function has validated the physiological relevance of these studies in detergent micelles. However, larger micelle and bicelle systems are sometimes required to stabilize the active forms of dynamic membrane proteins, such as the bacterial small multidrug resistance transporters. Even in these systems with aggregate molecular weights well over 100 kDa, solution NMR structural studies are feasible – but challenging. PMID:17961504

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

    Directory of Open Access Journals (Sweden)

    Erik Ronzone

    Full Text Available 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.

  8. Engineering hybrid exosomes by membrane fusion with liposomes

    Science.gov (United States)

    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

  9. Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

    Science.gov (United States)

    Ramakrishnan, N.; Sunil Kumar, P. B.; Radhakrishnan, Ravi

    2014-10-01

    Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham-Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the

  10. Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

    International Nuclear Information System (INIS)

    Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein–lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham–Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description

  11. Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

    Energy Technology Data Exchange (ETDEWEB)

    Ramakrishnan, N., E-mail: ramn@seas.upenn.edu [Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA-19104 (United States); Department of Bioengineering, University of Pennsylvania, Philadelphia, PA-19104 (United States); Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA-19104 (United States); Sunil Kumar, P.B., E-mail: sunil@physics.iitm.ac.in [Department of Physics, Indian Institute of Technology Madras, Chennai, 600036 (India); Radhakrishnan, Ravi, E-mail: rradhak@seas.upenn.edu [Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, PA-19104 (United States); Department of Bioengineering, University of Pennsylvania, Philadelphia, PA-19104 (United States); Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA-19104 (United States)

    2014-10-01

    Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein–lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham–Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description

  12. Mesoscale computational studies of membrane bilayer remodeling by curvature-inducing proteins

    Science.gov (United States)

    Ramakrishnan, N.; Sunil Kumar, P. B.; Radhakrishnan, Ravi

    2014-01-01

    Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham - Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this

  13. The structural role of cholesterol in cell membranes: from condensed bilayers to lipid rafts.

    Science.gov (United States)

    Krause, Martin R; Regen, Steven L

    2014-12-16

    CONSPECTUS: Defining the two-dimensional structure of cell membranes represents one of the most daunting challenges currently facing chemists, biochemists, and biophysicists. In particular, the time-averaged lateral organization of the lipids and proteins that make up these natural enclosures has yet to be established. As the classic Singer-Nicolson model of cell membranes has evolved over the past 40 years, special attention has focused on the structural role played by cholesterol, a key component that represents ca. 30% of the total lipids that are present. Despite extensive studies with model membranes, two fundamental issues have remained a mystery: (i) the mechanism by which cholesterol condenses low-melting lipids by uncoiling their acyl chains and (ii) the thermodynamics of the interaction between cholesterol and high- and low-melting lipids. The latter bears directly on one of the most popular notions in modern cell biology, that is, the lipid raft hypothesis, whereby cholesterol is thought to combine with high-melting lipids to form "lipid rafts" that float in a "sea" of low-melting lipids. In this Account, we first describe a chemical approach that we have developed in our laboratories that has allowed us to quantify the interactions between exchangeable mimics of cholesterol and low- and high-melting lipids in model membranes. In essence, this "nearest-neighbor recognition" (NNR) method involves the synthesis of dimeric forms of these lipids that contain a disulfide moiety as a linker. By means of thiolate-disulfide interchange reactions, equilibrium mixtures of dimers are then formed. These exchange reactions are initiated either by adding dithiothreitol to a liposomal dispersion to generate a small amount of thiol monomer or by including a small amount of thiol monomer in the liposomes at pH 5.0 and then raising the pH to 7.4. We then show how such NNR measurements have allowed us to distinguish between two very different mechanisms that have been

  14. Structure and dynamics of water and lipid molecules in charged anionic DMPG lipid bilayer membranes

    DEFF Research Database (Denmark)

    Rønnest, A. K.; Peters, Günther H.J.; Hansen, Flemming Yssing;

    2016-01-01

    phase with a monovalent counter-ion and in the gel phase with a divalent counter-ion. The diffusion constant of water as a function of its depth in the membrane has been determined from mean-square-displacement calculations. Also, calculated incoherent quasielastic neutron scattering functions have been...... compared to experimental results and used to determine an average diffusion constant for all water molecules in the system. On extrapolating the diffusion constants inferred experimentally to a temperature of 310 K, reasonable agreement with the simulations is obtained. However, the experiments do not have...... the sensitivity to confirm the diffusion of a small component of water bound to the lipids as found in the simulations. In addition, the orientation of the dipole moment of the water molecules has been determined as a function of their depth in the membrane. Previous indirect estimates of the...

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

    OpenAIRE

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

    2012-01-01

    Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered ...

  16. Porous Nanoparticle Supported Lipid Bilayers (Protocells) as Delivery Vehicles

    OpenAIRE

    Liu, Juewen; Stace-Naughton, Alison; Jiang, Xingmao; Brinker, C. Jeffrey

    2009-01-01

    Mixing liposomes with hydrophilic particles will induce fusion of the liposome onto the particle surface. Such supported bilayers have been extensively studied as a model for the cell membrane, while its application in drug delivery has not been pursued. In this communication, we report the use of phospholipids to achieve synergistic loading and encapsulating of a fluorescent dye (calcein) in mesoporous silica nanoparticles, and its delivery into mammalian cells. We found that cationic lipid ...

  17. From biological membranes to biomimetic model membranes

    Directory of Open Access Journals (Sweden)

    Eeman, M.

    2010-01-01

    Full Text Available Biological membranes play an essential role in the cellular protection as well as in the control and the transport of nutrients. Many mechanisms such as molecular recognition, enzymatic catalysis, cellular adhesion and membrane fusion take place into the biological membranes. In 1972, Singer et al. provided a membrane model, called fluid mosaic model, in which each leaflet of the bilayer is formed by a homogeneous environment of lipids in a fluid state including globular assembling of proteins and glycoproteins. Since its conception in 1972, many developments were brought to this model in terms of composition and molecular organization. The main development of the fluid mosaic model was made by Simons et al. (1997 and Brown et al. (1997 who suggested that membrane lipids are organized into lateral microdomains (or lipid rafts with a specific composition and a molecular dynamic that are different to the composition and the dynamic of the surrounding liquid crystalline phase. The discovery of a phase separation in the plane of the membrane has induced an explosion in the research efforts related to the biology of cell membranes but also in the development of new technologies for the study of these biological systems. Due to the high complexity of biological membranes and in order to investigate the biological processes that occur on the membrane surface or within the membrane lipid bilayer, a large number of studies are performed using biomimicking model membranes. This paper aims at revisiting the fundamental properties of biological membranes in terms of membrane composition, membrane dynamic and molecular organization, as well as at describing the most common biomimicking models that are frequently used for investigating biological processes such as membrane fusion, membrane trafficking, pore formation as well as membrane interactions at a molecular level.

  18. Membrane Requirement for Folding of the Herpes Simplex Virus 1 gB Cytodomain Suggests a Unique Mechanism of Fusion Regulation

    OpenAIRE

    Silverman, Jessica L.; Greene, Neil G.; King, David S.; Heldwein, Ekaterina E.

    2012-01-01

    Herpes simplex virus type 1 (HSV-1) enters cells by fusion of its envelope with a host cell membrane, which requires four viral glycoproteins and a cellular receptor. Viral fusion glycoprotein B (gB) mediates membrane fusion through the action of its ectodomain, while its cytoplasmic domain (cytodomain) regulates fusion from the opposite face of the membrane by an unknown mechanism. The gB cytodomain appears to restrict fusion, because point or truncation mutations within it increase the exte...

  19. Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

    International Nuclear Information System (INIS)

    4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellata root extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO4/H2O2, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO4/H2O2, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO4/H2O2. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation

  20. Antioxidant effect of 4-nerolidylcatechol and α-tocopherol in erythrocyte ghost membranes and phospholipid bilayers

    Energy Technology Data Exchange (ETDEWEB)

    Fernandes, K.S.; Silva, A.H.M.; Mendanha, S.A. [Instituto de Física, Universidade Federal de Goiás, Goiânia, GO (Brazil); Rezende, K.R. [Laboratório de Biofarmácia e Farmacocinética de Substâncias Bioativas, Faculdade de Farmácia, Universidade Federal de Goiás, Goiânia, GO (Brazil); Alonso, A. [Instituto de Física, Universidade Federal de Goiás, Goiânia, GO (Brazil)

    2013-09-06

    4-Nerolidylcatechol (4-NC) is found in Pothomorphe umbellata root extracts and is reported to have a topical protective effect against UVB radiation-induced skin damage, toxicity in melanoma cell lines, and antimalarial activity. We report a comparative study of the antioxidant activity of 4-NC and α-tocopherol against lipid peroxidation initiated by two free radical-generating systems: 2,2′-azobis(2-aminopropane) hydrochloride (AAPH) and FeSO{sub 4}/H{sub 2}O{sub 2}, in red blood cell ghost membranes and in egg phosphatidylcholine (PC) vesicles. Lipid peroxidation was monitored by membrane fluidity changes assessed by electron paramagnetic resonance spectroscopy of a spin-labeled lipid and by the formation of thiobarbituric acid-reactive substances. When lipoperoxidation was initiated by the hydroxyl radical in erythrocyte ghost membranes, both 4-NC and α-tocopherol acted in a very efficient manner. However, lower activities were observed when lipoperoxidation was initiated by the peroxyl radical; and, in this case, the protective effect of α-tocopherol was lower than that of 4-NC. In egg PC vesicles, malondialdehyde formation indicated that 4-NC was effective against lipoperoxidation initiated by both AAPH and FeSO{sub 4}/H{sub 2}O{sub 2}, whereas α-tocopherol was less efficient in protecting against lipoperoxidation by AAPH, and behaved as a pro-oxidant for FeSO{sub 4}/H{sub 2}O{sub 2}. The DPPH (2,2-diphenyl-1-picrylhydrazyl) free-radical assay indicated that two free radicals were scavenged per 4-NC molecule, and one free radical was scavenged per α-tocopherol molecule. These data provide new insights into the antioxidant capacity of 4-NC, which may have therapeutic applications for formulations designed to protect the skin from sunlight irradiation.

  1. Manipulating lipid bilayer material properties using biologically active amphipathic molecules

    Energy Technology Data Exchange (ETDEWEB)

    Ashrafuzzaman, Md [Department of Physiology and Biophysics, Weill Medical College of University of Cornell, New York, NY 10021 (United States); Lampson, M A [Department of Physiology and Biophysics, Weill Medical College of University of Cornell, New York, NY 10021 (United States); Greathouse, D V [Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701 (United States); II, R E Koeppe [Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701 (United States); Andersen, O S [Department of Physiology and Biophysics, Weill Medical College of University of Cornell, New York, NY 10021 (United States)

    2006-07-19

    Lipid bilayers are elastic bodies with properties that can be manipulated/controlled by the adsorption of amphipathic molecules. The resulting changes in bilayer elasticity have been shown to regulate integral membrane protein function. To further understand the amphiphile-induced modulation of bilayer material properties (thickness, intrinsic monolayer curvature and elastic moduli), we examined how an enantiomeric pair of viral anti-fusion peptides (AFPs)-Z-Gly-D-Phe and Z-Gly-Phe, where Z denotes a benzyloxycarbonyl group, as well as Z-Phe-Tyr and Z-D-Phe-Phe-Gly-alters the function of enantiomeric pairs of gramicidin channels of different lengths in planar bilayers. For both short and long channels, the channel lifetimes and appearance frequencies increase as linear functions of the aqueous AFP concentration, with no apparent effect on the single-channel conductance. These changes in channel function do not depend on the chirality of the channels or the AFPs. At pH 7.0, the relative changes in channel lifetimes do not vary when the channel length is varied, indicating that these compounds exert their effects primarily by causing a positive-going change in the intrinsic monolayer curvature. At pH 4.0, the AFPs are more potent than at pH 7.0 and have greater effects on the shorter channels, indicating that these compounds now change the bilayer elastic moduli. When AFPs of different anti-fusion potencies are compared, the rank order of the anti-fusion activity and the channel-modifying activity is similar, but the relative changes in anti-fusion potency are larger than the changes in channel-modifying activity. We conclude that gramicidin channels are useful as molecular force transducers to probe the influence of small amphiphiles upon lipid bilayer material properties.

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

    Science.gov (United States)

    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.

  3. Interaction of antimicrobial peptide Plantaricin149a and four analogs with lipid bilayers and bacterial membranes

    Directory of Open Access Journals (Sweden)

    José Luiz de Souza Lopes

    2013-12-01

    Full Text Available The amidated analog of Plantaricin149, an antimicrobial peptide from Lactobacillus plantarum NRIC 149, directly interacts with negatively charged liposomes and bacterial membranes, leading to their lysis. In this study, four Pln149-analogs were synthesized with different hydrophobic groups at their N-terminus with the goal of evaluating the effect of the modifications at this region in the peptide's antimicrobial properties. The interaction of these peptides with membrane models, surface activity, their hemolytic effect on red blood cells, and antibacterial activity against microorganisms were evaluated. The analogs presented similar action of Plantaricin149a; three of them with no hemolytic effect (< 5% until 0.5 mM, in addition to the induction of a helical element when binding to negative liposomes. The N-terminus difference between the analogs and Plantaricin149a retained the antibacterial effect on S. aureus and P. aeruginosa for all peptides (MIC50 of 19 µM and 155 µM to Plantaricin149a, respectively but resulted in a different mechanism of action against the microorganisms, that was bactericidal for Plantaricin149a and bacteriostatic for the analogs. This difference was confirmed by a reduction in leakage action for the analogs. The lytic activity of Plantaricin149a is suggested to be a result of the peptide-lipid interactions from the amphipathic helix and the hydrophobic residues at the N-terminus of the antimicrobial peptide.

  4. Membrane support of accelerated fuel capsules for inertial fusion energy reactors

    International Nuclear Information System (INIS)

    The use of a thin membrane to suspend an (inertial fusion energy) fuel capsule in a holder for injection into a reactor chamber is investigated. Capsule displacement and membrane deformation angle are calculated for an axisymmetric geometry for a range of membrane strain and capsule size. This information is used to calculate maximum target accelerations. Membranes must be thin (perhaps of order one micron) to minimize their effect on capsule implosion symmetry. For example, a 5 μm thick cryogenic mylar membrane is calculated to allow 1,000 m/s2 acceleration of a 3 mm radius, 100 mg capsule. Vibration analysis (for a single membrane support) shows that if membrane vibration is not deliberately minimized, allowed acceleration may be reduced by a factor of four. A two membrane alternative geometry would allow several times greater acceleration. Therefore, alternative membrane geometry's should be used to provide greater target acceleration potential and reduce capsule displacement within the holder (for a given membrane thickness)

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

    International Nuclear Information System (INIS)

    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

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

    Science.gov (United States)

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

    2016-02-23

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

  7. Docosahexaenoic acid and eicosapentaenoic acid induce changes in the physical properties of a lipid bilayer model membrane.

    Science.gov (United States)

    Onuki, Yoshinori; Morishita, Mariko; Chiba, Yoshiyuki; Tokiwa, Shinji; Takayama, Kozo

    2006-01-01

    We investigated the effect of fatty acids such as stearic acid (SA, 18:0), oleic acid (OA, 18:1), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6) on a dipalmitoylphosphatidylcholine (DPPC) bilayer by determining the phase transition temperature, fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), and detergent insolubility. Treatment with unsaturated fatty acid broadened and shifted the phase transitions of the DPPC bilayer to a lower temperature. The phase transition temperature and the value of fluorescence anisotropy of DPH at 37 degrees C decreased progressively with increasing treatment amounts of unsaturated fatty acid. A large amount of the DPPC bilayer treated with unsaturated fatty acid was dissolved in Triton X-100, obtaining a low level of detergent insolubility. These modifications of the bilayer physical properties were most pronounced with DHA and EPA treatment. These data show that unsaturated fatty acids, particularly DHA and EPA, induce a marked change in the lipid bilayer structure. The composition of fatty acids in the DPPC bilayer was similar after treatment with various unsaturated fatty acids, suggesting that the different actions of unsaturated fatty acids are attributed to change in the molecular structure (e.g., kinked conformation by double bonds). We further explored the change in physical properties induced by fatty acids dispersed in a water-in-oil-in-water multiple emulsion and found that unsaturated fatty acids acted efficiently on the DPPC bilayer, even when incorporated in emulsion form. PMID:16394552

  8. β2 Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

    Science.gov (United States)

    Bubnell, Jaclyn; Pfister, Patrick; Sapar, Maria L.; Rogers, Matthew E.; Feinstein, Paul

    2013-01-01

    Green fluorescent protein (GFP) has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs), Cerulean (and mCerulean3), Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR). We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β2AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality. PMID:24086401

  9. Diffusion studies on permeable nitroxyl spin probe through lipid bilayer membrane

    Energy Technology Data Exchange (ETDEWEB)

    Benial, A. Milton Franklin; Meenakumari, V. [Department of Physics, NMSSVN College, Nagamalai, Madurai-625019 (India); Ichikawa, Kazuhiro; Yamada, Ken-ichi; Utsumi, Hideo, E-mail: hideo.utsumi.278@m.kyushu-u.ac.jp [Department of Bio-functional Science, Kyushu University, Fukuoka (Japan); Hyodo, Fuminori [Innovation Center for Medical Redox Navigation, Kyushu University, Fukuoka (Japan); Jawahar, A. [Department of Chemistry, NMSSVN College, Nagamalai, Madurai-625 019 (India)

    2014-04-24

    Electron spin resonance (ESR) studies were carried out for 2mM {sup 14}N labeled deutrated permeable 3- methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (MC-PROXYL) in pure water, 1 mM, 2 mM, 3 mM and 4 mM concentration of MC-PROXYL in 300 mM concentration of liposomal solution by using a L-band ESR spectrometer. The ESR parameters such as linewidth, hyperfine coupling constant, g-factor, partition parameter and permeability were reported. The partition parameter and permeability values indicate the maximum spin distribution in the lipid phase at 2 mM concentration. This study illustrates that ESR can be used to differentiate between the intra and extra-membrane water by loading the liposome vesicles with a lipid-permeable nitroxyl spin probe. From the ESR results, the radical concentration was optimized as 2 mM in liposomal solution for ESR phantom studies and experiments.

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

    International Nuclear Information System (INIS)

    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 125I-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

  11. Amino Acid Sequence Requirements of the Transmembrane and Cytoplasmic Domains of Influenza Virus Hemagglutinin for Viable Membrane Fusion

    OpenAIRE

    Melikyan, Grigory B.; Lin, Sasa; Roth, Michael G.; Cohen, Fredric S.

    1999-01-01

    The amino acid sequence requirements of the transmembrane (TM) domain and cytoplasmic tail (CT) of the hemagglutinin (HA) of influenza virus in membrane fusion have been investigated. Fusion properties of wild-type HA were compared with those of chimeras consisting of the ectodomain of HA and the TM domain and/or CT of polyimmunoglobulin receptor, a nonviral integral membrane protein. The presence of a CT was not required for fusion. But when a TM domain and CT were present, fusion activity w...

  12. Anomalous viscosity effect in the early stages of the ion-assisted adhesion/fusion event between lipid bilayers: A theoretical and computational study

    OpenAIRE

    Raudino, Antonio; Marrink, Siewert J.; Pannuzzo, Martina

    2013-01-01

    The effect of viscosity on the encounter rate of two interacting membranes was investigated by combining a non-equilibrium Fokker-Planck model together with extensive Molecular Dynamics (MD) calculations. The encounter probability and stabilization of transient contact points represent the preliminary steps toward short-range adhesion and fusion of lipid leaflets. To strengthen our analytical model, we used a Coarse Grained MD method to follow the behavior of two charged palmitoyl oleoyl phos...

  13. Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization

    OpenAIRE

    Christian Kleusch; Bernd Hoffmann; Nils Hersch; Agnes Csiszár; Rudolf Merkel

    2012-01-01

    In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traf...

  14. Phospholipid bilayers are viscoelastic

    OpenAIRE

    Harland, Christopher W.; Bradley, Miranda J.; Parthasarathy, Raghuveer

    2010-01-01

    Lipid bilayers provide the structural framework for cellular membranes, and their character as two-dimensional fluids enables the mobility of membrane macromolecules. Though the existence of membrane fluidity is well established, the nature of this fluidity remains poorly characterized. Three-dimensional fluids as diverse as chocolates and cytoskeletal networks show a rich variety of Newtonian and non-Newtonian dynamics that have been illuminated by contemporary rheological techniques. Applyi...

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

    OpenAIRE

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

    2013-01-01

    Author Summary Many viruses package their genetic material into a lipid envelope. In order to deliver their genome into the host-cell cytoplasm, where it can be replicated, viruses must fuse their envelope with a cellular lipid membrane. This fusion event is therefore a critical step in the entry of an enveloped virus into the cell. In this study, we used various cell biological and biochemical approaches to map precisely the cell entry pathway of two major human pathogens from the flavivirus...

  16. A molecular model for membrane fusion based on solution studies of an amphiphilic peptide from HIV gp41.

    OpenAIRE

    Fujii, G; Horvath, S.; Woodward, S.; Eiserling, F.; Eisenberg, D.

    1992-01-01

    The mechanism of protein-mediated membrane fusion and lysis has been investigated by solution-state studies of the effects of peptides on liposomes. A peptide (SI) corresponding to a highly amphiphilic C-terminal segment from the envelope protein (gp41) of the human immunodeficiency virus (HIV) was synthesized and tested for its ability to cause lipid membranes to fuse together (fusion) or to break open (lysis). These effects were compared to those produced by the lytic and fusogenic peptide ...

  17. Two Coiled-Coil Domains of Chlamydia trachomatis IncA Affect Membrane Fusion Events during Infection

    OpenAIRE

    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-me...

  18. A GALA lipopeptide mediates pH- and membrane charge dependent fusion with stable giant unilamellar vesicles

    DEFF Research Database (Denmark)

    Etzerodt, Thomas P.; Trier, Sofie; Henriksen, Jonas R.; Andresen, Thomas L.

    2012-01-01

    Peptides capable of mediating fusion between lipid membranes are widely observed in nature, and have attracted considerable attention in the liposome drug delivery field. However, studies that are proving the benefit of small synthetic fusion peptides as components in drug delivery systems remain...

  19. Septins promote macropinosome maturation and traffic to the lysosome by facilitating membrane fusion.

    Science.gov (United States)

    Dolat, Lee; Spiliotis, Elias T

    2016-08-29

    Macropinocytosis, the internalization of extracellular fluid and material by plasma membrane ruffles, is critical for antigen presentation, cell metabolism, and signaling. Macropinosomes mature through homotypic and heterotypic fusion with endosomes and ultimately merge with lysosomes. The molecular underpinnings of this clathrin-independent endocytic pathway are largely unknown. Here, we show that the filamentous septin GTPases associate preferentially with maturing macropinosomes in a phosphatidylinositol 3,5-bisphosphate-dependent manner and localize to their contact/fusion sites with macropinosomes/endosomes. Septin knockdown results in large clusters of docked macropinosomes, which persist longer and exhibit fewer fusion events. Septin depletion and overexpression down-regulates and enhances, respectively, the delivery of fluid-phase cargo to lysosomes, without affecting Rab5 and Rab7 recruitment to macropinosomes/endosomes. In vitro reconstitution assays show that fusion of macropinosomes/endosomes is abrogated by septin immunodepletion and function-blocking antibodies and is induced by recombinant septins in the absence of cytosol and polymerized actin. Thus, septins regulate fluid-phase cargo traffic to lysosomes by promoting macropinosome maturation and fusion with endosomes/lysosomes. PMID:27551056

  20. Microfluidic anodization of aluminum films for the fabrication of nanoporous lipid bilayer support structures

    Science.gov (United States)

    Bhattacharya, Jaydeep; Kisner, Alexandre; Offenhäusser, Andreas

    2011-01-01

    Summary Solid state nanoporous membranes show great potential as support structures for biointerfaces. In this paper, we present a technique for fabricating nanoporous alumina membranes under constant-flow conditions in a microfluidic environment. This approach allows the direct integration of the fabrication process into a microfluidic setup for performing biological experiments without the need to transfer the brittle nanoporous material. We demonstrate this technique by using the same microfluidic system for membrane fabrication and subsequent liposome fusion onto the nanoporous support structure. The resulting bilayer formation is monitored by impedance spectroscopy across the nanoporous alumina membrane in real-time. Our approach offers a simple and efficient methodology to investigate the activity of transmembrane proteins or ion diffusion across membrane bilayers. PMID:21977420

  1. A molecular basis explanation of the dynamic and thermal effects of vinblastine sulfate upon dipalmitoylphosphatidylcholine bilayer membranes.

    Science.gov (United States)

    Maswadeh, H; Demetzos, C; Daliani, I; Kyrikou, I; Mavromoustakos, T; Tsortos, A; Nounesis, G

    2002-12-23

    Differential scanning calorimetry has been employed to study the thermal effects of vinblastine sulfate upon aqueous, single and multiple bilayer dispersions of 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC). The calorimetric results summarized to an increase in the gel to liquid-crystalline phase transition enthalpy and the abolishment of the L(beta)' (gel phase) to P(beta)' (ripple phase) pretransition for the uni- and multilamellar dispersions, as well as an increase in the transition temperature T(m) and the transition cooperativity for single bilayer DPPC/vinblastine mixed vesicles, are consistent with an induced, partially interdigitated, gel phase. Computational analysis has been successfully applied to clarify the intermolecular effects and verify the feasibility of the proposed interdigitation for the vinblastine sulfate molecules and also for the ursodeoxycholic acid (UDCAH) and bromocylated taxanes, which have been shown to induce an interdigitated gel phase in DPPC bilayers. PMID:12488037

  2. The temperature-dependent physical state of polar lipids and their miscibility impact the topography and mechanical properties of bilayer models of the milk fat globule membrane.

    Science.gov (United States)

    Murthy, Appala Venkata Ramana; Guyomarc'h, Fanny; Lopez, Christelle

    2016-09-01

    The polar lipid assembly and biophysical properties of the biological membrane enveloping the milk fat globules (the MFGM) are yet poorly known, especially in connection with the temperature history that milk can experience after its secretion. However, bioactive mechanisms depend on biological structure, which itself highly depend on temperature. The objectives of this study were to investigate polar lipid packing in hydrated bilayers, models of the MFGM, and to follow at intermolecular level temperature-induced changes in the range 60-6°C, using the combination of differential scanning calorimetry, X-ray diffraction, atomic force microscopy (AFM) imaging and force spectroscopy. MFGM polar lipids, especially sphingomyelin, contain long chain saturated fatty acids with high phase transition temperatures. On cooling, the liquid disordered ld to solid ordered so (gel) phase transition of MFGM polar lipids started at about 40°C, leading to phase separation and formation of so phase domains protruding by about 1nm from the ld phase. Indentation measurements using AFM revealed that the resistance of the so phase domains to rupture was significantly higher than that of the ld phase and that it increased for both the domain and fluid phases with decreasing temperature. However, packing and stability of the bilayers were adversely affected by fast cooling to 6°C or by cooling-rewarming cycle. This study showed that MFGM polar lipid bilayers are dynamic systems. Heterogeneity in the structure and mechanical properties of the membrane was induced by temperature-dependent so/ld phase immiscibility of the lipid components. This could have consequences on the MFGM technological and biological functions (e.g. immunity and milk lipid digestion). PMID:27349732

  3. Oxygen as a paramagnetic probe for nuclear magnetic resonance: structure and paramagnetic profile of a lipid bilayer/membrane model system

    International Nuclear Information System (INIS)

    Paramagnetic contact shifts and relaxation rate enhancements from molecular oxygen dissolved in a model membrane, were studied by nuclear magnetic resonance spectroscopy. The model membrane system was an isotropic bicelle formed using 1-myristelaidoyl-2-myristoyl-d27-sn- glycero-3-phosphocholine (MLMPC), a custom phospholipid, and 1-2-dihexanoyl-d22-sn-glycero-3-phosphocholine (DHPC). The 13C and 1H spectra of MLMPC were assigned. Molecular oxygen was delivered at external pressures of 20 and 50 atm. Paramagnetic contact shifts were found to scale with the oxygen solubility gradient in the lipid bilayer, were found to be invariant to temperature changes in the region studied (288K to 331K), and scaled linearly with changes in oxygen pressure. Relaxation rate enhancements from oxygen were low in the headgroup region and increased to a roughly constant rate in the acyl chain region. Rates were comparable to values predicted by simple thermodynamic theories which take into account the observed gradients in diffusion rates and solubility of oxygen in bilayers. (author)

  4. Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

    Directory of Open Access Journals (Sweden)

    Bárbara Hissa

    Full Text Available 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

  5. Study on a multi-component palladium alloy membrane for the fusion fuel cleanup system

    International Nuclear Information System (INIS)

    Demonstration Tests with (D,T)2 gas to examine the reported hydrogen embrittlement and helium damage on Pd and Pd-Ag binary alloy are needed for a palladium alloy membrane for its application to a fusion fuel system. T2-gas circulating and T2-gas immersion tests with a multi-component palladium alloy, which had been selected for use of tritum purification, have been performed in the Tritium Systems Test Assembly(TSTA) at Los Alamos National Laboratory under the Japan/US Fusion Cooperation Program. Mechanical tensile tests and metallographic studies have been conducted in these durability tests. Similar tests had been performed on the same material under tritium-free atmospheres(H2, N2) to analyse the data obtained by the T2-gas tests. This report describes the results of the mechanical tensile tests and the test conditions. (author)

  6. Ebola virus glycoprotein needs an additional trigger, beyond proteolytic priming for membrane fusion.

    Directory of Open Access Journals (Sweden)

    Shridhar Bale

    2011-11-01

    Full Text Available BACKGROUND: Ebolavirus belongs to the family filoviridae and causes severe hemorrhagic fever in humans with 50-90% lethality. Detailed understanding of how the viruses attach to and enter new host cells is critical to development of medical interventions. The virus displays a trimeric glycoprotein (GP(1,2 on its surface that is solely responsible for membrane attachment, virus internalization and fusion. GP(1,2 is expressed as a single peptide and is cleaved by furin in the host cells to yield two disulphide-linked fragments termed GP1 and GP2 that remain associated in a GP(1,2 trimeric, viral surface spike. After entry into host endosomes, GP(1,2 is enzymatically cleaved by endosomal cathepsins B and L, a necessary step in infection. However, the functional effects of the cleavage on the glycoprotein are unknown. PRINCIPAL FINDINGS: We demonstrate by antibody binding and Hydrogen-Deuterium Exchange Mass Spectrometry (DXMS of glycoproteins from two different ebolaviruses that although enzymatic priming of GP(1,2 is required for fusion, the priming itself does not initiate the required conformational changes in the ectodomain of GP(1,2. Further, ELISA binding data of primed GP(1,2 to conformational antibody KZ52 suggests that the low pH inside the endosomes also does not trigger dissociation of GP1 from GP2 to effect membrane fusion. SIGNIFICANCE: The results reveal that the ebolavirus GP(1,2 ectodomain remains in the prefusion conformation upon enzymatic cleavage in low pH and removal of the glycan cap. The results also suggest that an additional endosomal trigger is necessary to induce the conformational changes in GP(1,2 and effect fusion. Identification of this trigger will provide further mechanistic insights into ebolavirus infection.

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

    International Nuclear Information System (INIS)

    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[125I] 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 F1 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 F1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and the F2 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

  8. Neutron diffraction studies of viral fusion peptides

    Science.gov (United States)

    Bradshaw, Jeremy P.; J. M. Darkes, Malcolm; Katsaras, John; Epand, Richard M.

    2000-03-01

    Membrane fusion plays a vital role in a large and diverse number of essential biological processes. Despite this fact, the precise molecular events that occur during fusion are still not known. We are currently engaged on a study of membrane fusion as mediated by viral fusion peptides. These peptides are the N-terminal regions of certain viral envelope proteins that mediate the process of fusion between the viral envelope and the membranes of the host cell during the infection process. As part of this study, we have carried out neutron diffraction measurements at the ILL, BeNSC and Chalk River, on a range of viral fusion peptides. The peptides, from simian immunodeficiency virus (SIV), influenza A and feline leukaemia virus (FeLV), were incorporated into stacked phospholipid bilayers. Some of the peptides had been specifically deuterated at key amino acids. Lamellar diffraction data were collected and analysed to yield information on the peptide conformation, location and orientation relative to the bilayer.

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

    Science.gov (United States)

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

    2016-02-01

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

  10. Preliminary design of fusion reactor fuel cleanup system by palladium alloy membrane method

    International Nuclear Information System (INIS)

    A design of palladium diffuser and Fuel Cleanup System (FCU) for D-T fusion reactor is proposed. Feasibility of palladium alloy membrane method is discussed based on the early studies by the authors. Operating conditions of the palladium diffuser are determined experimentally. Dimensions of the diffuser are estimated from computer simulation. FCU system is designed under the feed conditions of Tritium Systems Test Assembly (TSTA) at Los Alamos Scientific Laboratory. The system is composed of Pd-diffusers, catalytic oxidizer, freezer and zink beds, and has some advantages in system layout and operation. This design can readily be extended to other conditions of plasma exhaust gases. (author)

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

    Science.gov (United States)

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

    2005-06-01

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

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

    Science.gov (United States)

    Majumdar, Anupa; Sarkar, Munna

    2016-06-01

    Biological membranes are made up of a variety of lipids with diverse physicochemical properties. The lipid composition modulates different lipidic parameters, such as hydration, dynamics, lipid packing, curvature strain, etc. Changes in these parameters affect various membrane-mediated processes, such as membrane fusion which is an integral step in many biological processes. Packing defects, which originate either from mismatch in the headgroup region or in the hydrophobic acyl tail region, play a major role in modulating membrane dynamics. In this study, we demonstrate how even a small mismatch in the fatty acyl chain length, achieved by incorporation of low concentrations (up to 30 mol %) of dipalmitoylphosphatidylcholine (DPPC) into dimyristoylphosphatidylcholine (DMPC) small unilamellar vesicles (SUVs), alters several lipidic parameters like packing, dynamics, and headgroup hydration. This in turn affects non steroidal anti-inflammatory drug (NSAID) induced membrane fusion. Dynamic light scattering, differential scanning calorimetry, second-derivative absorption spectrophotometry, and steady-state and time-resolved fluorescence have been used to elucidate the effect of small mismatch in the tails in DMPC/DPPC mixed vesicles and how it modulates membrane fusion induced by the oxicam NSAIDs, meloxicam (Mx), piroxicam (Px), and tenoxicam (Tx). Fusion kinetics was monitored using fluorescence based fusion assays. At low DPPC concentration of 10 mol %, additional fluidization promotes lipid mixing to some extent for Mx, but at higher mol % of DPPC, subsequent increase in rigidity of membrane interior along with increase in headgroup hydration, synergistically inhibits fusion to various extents for the three different drugs, Mx, Px, and Tx. PMID:27153337

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

    Directory of Open Access Journals (Sweden)

    Jian Wu

    2015-01-01

    Full Text Available 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.

  14. Membrane separation technologies: their application to the fusion reactor fuel cycle

    International Nuclear Information System (INIS)

    The future fusion reactor fuel will be a mixture of deuterium and tritium. Deuterium is produced using traditional separation technology. Tritium, on the contrary, must be produced by means of a nuclear reaction between neutrons and lithium atoms within the reactor breeder which could be a lithiated ceramic material or a liquid metal containing lithium. The tritium produced in the breeder needs a proper extraction process to reach the required purity level. A conceptual modified version of the tritium recovery plant for a solid ceramic breeder, working with two membranes reaction/separation units, is studied in this work. The first considered membrane unit is a catalytic ceramic membrane reactor to remove, via oxidation, the hydrogen isotopes from the purge gas (He); the second is a Pd/Ag membrane permeator to separate the hydrogen isotopes from the water shift reactor gaseous products. The modelling and the mass balances have been obtained either on the basis of data in the literature or on experimental results. (orig.)

  15. A neutron study of the feline leukaemia virus fusion peptide: Implications for biological fusion?

    Science.gov (United States)

    Davies, Sarah M. A.; Darkes, Malcolm J. M.; Bradshaw, Jeremy P.

    Neutron diffraction studies were performed on stacked phospholipid bilayers to determine the effects of the feline leukaemia virus (FeLV) fusion peptide on membrane structure. Bilayers were composed of dioleoylphosphatidylcholine with 50% (mol) dioleoylphosphatidylglycerol. Neutron scattering profiles with peptide present showed an increase in scattering density in the lipid-tails region, whilst scattering by the lipid headgroup region was decreased. This is interpreted as a lowering of the packing density of the lipid headgroups and an increase in the packing density of the lipid tails. Modelling studies and experimental evidence have suggested that fusion peptides catalyse fusion by increasing the negative curvature of the target membrane's outer monolayer. Our results presented here add support to this hypothesis for the fusion mechanism. The 2H 2O scattering profile was also slightly perturbed in the lipid headgroup region with 1% (mol)FeLV fusion peptide present. The FeLV peptide had no significant effect on the organisation of bilayers containing only dioleoylphosphatidylcholine.

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

    International Nuclear Information System (INIS)

    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. Open channel current noise analysis of S6 peptides from KvAP channel on bilayer lipid membrane shows bimodal power law scaling

    Science.gov (United States)

    Shrivastava, Rajan; Malik, Chetan; Ghosh, Subhendu

    2016-06-01

    Open channel current noise in synthetic peptide S6 of KvAP channel was investigated in a voltage clamp experiment on bilayer lipid membrane (BLM). It was observed that the power spectral density (PSD) of the component frequencies follows power law with different slopes in different frequency ranges. In order to know the origin of the slopes PSD analysis was done with signal filtering. It was found that the first slope in the noise profile follows 1 / f pattern which exists at lower frequencies and has high amplitude current noise, while the second slope corresponds to 1 /f 2 - 3 pattern which exists at higher frequencies with low amplitude current noise. In addition, white noise was observed at very large frequencies. It was concluded that the plausible reason for the multiple power-law scaling is the existence of different modes of non-equilibrium ion transport through the S6 channel.

  18. Identification and characterization of LFD-2, a predicted fringe protein required for membrane integrity during cell fusion in neurospora crassa.

    Science.gov (United States)

    Palma-Guerrero, Javier; Zhao, Jiuhai; Gonçalves, A Pedro; Starr, Trevor L; Glass, N Louise

    2015-03-01

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

  19. Fusion

    CERN Document Server

    Mahaffey, James A

    2012-01-01

    As energy problems of the world grow, work toward fusion power continues at a greater pace than ever before. The topic of fusion is one that is often met with the most recognition and interest in the nuclear power arena. Written in clear and jargon-free prose, Fusion explores the big bang of creation to the blackout death of worn-out stars. A brief history of fusion research, beginning with the first tentative theories in the early 20th century, is also discussed, as well as the race for fusion power. This brand-new, full-color resource examines the various programs currently being funded or p

  20. Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Mote, Kaustubh R. [University of Minnesota, Department of Chemistry (United States); Gopinath, T. [University of Minnesota, Department of Biochemistry, Molecular Biology and Biophysics (United States); Veglia, Gianluigi, E-mail: vegli001@umn.edu [University of Minnesota, Department of Chemistry (United States)

    2013-10-15

    The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD {approx}0.44 A, a tilt angle of 24 Degree-Sign {+-} 1 Degree-Sign , and an azimuthal angle of 55 Degree-Sign {+-} 6 Degree-Sign . This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR.

  1. Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

    International Nuclear Information System (INIS)

    The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD ∼0.44 Å, a tilt angle of 24° ± 1°, and an azimuthal angle of 55° ± 6°. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR

  2. Computer Simulations of Lipid Bilayers and Proteins

    DEFF Research Database (Denmark)

    Sonne, Jacob

    2006-01-01

    The importance of computer simulations in lipid bilayer research has become more prominent for the last couple of decades and as computers get even faster, simulations will play an increasingly important part of understanding the processes that take place in and across cell membranes. This thesis...... entitled Computer simulations of lipid bilayers and proteins describes two molecular dynamics (MD) simulation studies of pure lipid bilayers as well as a study of a transmembrane protein embedded in a lipid bilayer matrix. Below follows a brief overview of the thesis. Chapter 1. This chapter is a short......, Pressure profile calculations in lipid bilayers: A lipid bilayer is merely $\\sim$5~nm thick, but the lateral pressure (parallel to the bilayer plane) varies several hundred bar on this short distance (normal to the bilayer). These variations in the lateral pressure are commonly referred to as the pressure...

  3. Molecular Dynamics Simulation of HIV Fusion Inhibitor T-1249: Insights on Peptide-Lipid Interaction

    Directory of Open Access Journals (Sweden)

    A. M. T. Martins do Canto

    2012-01-01

    Full Text Available T-1249 is a peptide that inhibits the fusion of HIV envelope with the target cell membrane. Recent results indicate that T-1249, as in the case of related inhibitor peptide T-20 (enfuvirtide, interacts with membranes, more extensively in the bilayer liquid disordered phase than in the liquid ordered state, which could be linked to its effectiveness. Extensive molecular dynamics simulations (100 ns were carried out to investigate the interaction between T-1249 and bilayers of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC and POPC/cholesterol (1 : 1. It was observed that T-1249 interacts to different extents with both membrane systems and that peptide interaction with the bilayer surface has a local effect on membrane structure. Formation of hydrogen bonding between certain peptide residues and several acceptor and donor groups in the bilayer molecules was observed. T-1249 showed higher extent of interaction with bilayers when compared to T-20. This is most notable in POPC/Chol membranes, owing to more peptide residues acting as H bond donors and acceptors between the peptide and the bilayer lipids, including H-bonds formed with cholesterol. This behavior is at variance with that of T-20, which forms no H bonds with cholesterol. This higher ability to interact with membranes is probably correlated with its higher inhibitory efficiency.

  4. Porous nanoparticle supported lipid bilayers (protocells) as delivery vehicles.

    Science.gov (United States)

    Liu, Juewen; Stace-Naughton, Alison; Jiang, Xingmao; Brinker, C Jeffrey

    2009-02-01

    Mixing liposomes with hydrophilic particles induces fusion of the liposome onto the particle surface. Such supported bilayers have been studied extensively as models of the cell membrane, while their applications in drug delivery have not been pursued. In this communication, we report liposome fusion on mesoporous particles as a synergistic means to simultaneously load and seal cargo within the porous core. We find fusion of a cationic lipid (DOTAP) on an anionic silica particle loads an anionic fluorescent dye (calcein) into the particle to a concentration exceeding 100x that in the surrounding medium. The loaded "protocell" particles are taken up efficiently by Chinese hamster ovary cells, where, due to a reduced pH within endosomal compartments, calcein is effectively released. Compared to some other nanoparticle systems, protocells provide a simple construct for cargo loading, sealing, delivery, and release. They promise to serve as useful vectors in nanomedicine. PMID:19173660

  5. Applications of superpermeable membranes in fusion: the flux density problem and experimental progress

    Energy Technology Data Exchange (ETDEWEB)

    Livshits, A. [Bonch-Bruyevich Univ., St. Petersburg (Russian Federation); Ohyabu, N. [National Institute for Fusion Science, Furoh-cho, Chikusa-ku, Nagoya 464-01 (Japan); Notkin, M. [Bonch-Bruyevich Univ., St. Petersburg (Russian Federation); Alimov, V. [Bonch-Bruyevich Univ., St. Petersburg (Russian Federation); Suzuki, H. [National Institute for Fusion Science, Furoh-cho, Chikusa-ku, Nagoya 464-01 (Japan); Samartsev, A. [Bonch-Bruyevich Univ., St. Petersburg (Russian Federation); Solovyov, M. [Bonch-Bruyevich Univ., St. Petersburg (Russian Federation); Grigoriadi, I. [Bonch-Bruyevich Univ., St. Petersburg (Russian Federation); Glebovsky, A. [Bonch-Bruyevich Univ., St. Petersburg (Russian Federation); Busnyuk, A. [Bonch-Bruyevich Univ., St. Petersburg (Russian Federation); Doroshin, A. [Bonch-Bruyevich Univ., St. Petersburg (Russian Federation); Komatsu, K. [National Institute for Fusion Science, Furoh-cho, Chikusa-ku, Nagoya 464-01 (Japan)

    1997-02-01

    Superpermeable membranes whose permeability to energetic hydrogen approaches the permeability of an opening of the same area can be employed to separate D/T and He in fusion machine exhausts, to control the edge plasma and divertor conditions (by pumping and/or arranging of gas circulation through SOL or divertor) and to pump and recuperate D/T in auxiliary systems e.g. in pellet or neutral beam injection. One of the key points is the operation at permeation flux densities of up to 10{sup 16}-10{sup 19} cm{sup -2} s{sup -1}. Theory predicts that the highest flux densities may be reached with superpermeable membranes based on the V group metals: the limit conditioned by a maximum permissible hydrogen concentration in bulk metal is expected to be as high as {proportional_to}10{sup 19} cm{sup -2} s{sup -1}. The experimental membrane system comprised a cylindrical Nb membrane and an incandescent Ta/Nb atomizer placed inside. The hydrogen pumping speed by this system amounts to {proportional_to}10{sup 3} l/s, with a specific pumping speed of {proportional_to}1 l/s per cm{sup 2} membrane area and {proportional_to}8 l/s per cm{sup 2} atomizer area. Superpermeability was observed at record parameters referring both to the flux density of 3 x 10{sup 17} H/cm{sup 2}/s (by one order of magnitude larger than ever before) and to the operational pressure of 3 x 10{sup -2} Torr. A long-term reliable operation of this system proved being possible even in a vacuum far inferior to UHV conditions. (orig.).

  6. Interaction of n-octyl β,D-glucopyranoside with giant magnetic-fluid-loaded phosphatidylcholine vesicles: direct visualization of membrane curvature fluctuations as a function of surfactant partitioning between water and lipid bilayer.

    Science.gov (United States)

    Ménager, Christine; Guemghar, Dihya; Cabuil, Valérie; Lesieur, Sylviane

    2010-10-01

    The present study deals with the morphological modifications of giant dioleoyl phosphatidylcholine vesicles (DOPC GUVs) induced by the nonionic surfactant n-octyl β,D-glucopyranoside at sublytic levels, i.e., in the first steps of the vesicle-to-micelle transition process, when surfactant inserts into the vesicle bilayer without disruption. Experimental conditions were perfected to exactly control the surfactant bilayer composition of the vesicles, in line with former work focused on the mechanical properties of the membrane of magnetic-fluid-loaded DOPC GUVs submitted to a magnetic field. The purpose here was to systematically examine, in the absence of any external mechanical constraint, the dynamics of giant vesicle shape and membrane deformations as a function of surfactant partitioning between the aqueous phase and the lipid membrane, beforehand established by turbidity measurements from small unilamellar vesicles. PMID:20825201

  7. Ion Channel Behavior of a Supported Bilayer Lipid Membrane Composed of 5,5-Ditetradecyl-2- (2-trimethyl-ammonioethyl)-l, 3-dioxane Bromide Modified Glassy Carbon Electrode

    Institute of Scientific and Technical Information of China (English)

    龚静鸣; 林祥钦

    2003-01-01

    A synthetic cationic surfactant, 5,5-ditetradecyl-2-(2-trimethyl-ammonioethyl)-l,3-dioxane bromide (DTDB), was used to construct a supported bilayer lipid membrane (s-BLM) coatedon an underlying glassy carbon electrode (GCE). Electrochemical impedance spectroscopy (EIS), small-angle X-ray diffraction (SAXD) and cyclic voltammetry were used to characterize the s-BLM. Both EIS and SAXD data indicated that the synthetic lipid exists as a well-oriented bilayer in the membrane.The voltammetric study showed that the lipid membrane can open ion channels in the presence of ClO4- stimulant with Ru(bpy)32+ as marker ions and give distinct channel currents.The channels can be dosed and open up again many times by removing or introducing ClO4- anions.

  8. Melittin induces HII phase formation in cardiolipin model membranes

    OpenAIRE

    Batenburg, A M; Hibbeln, J.C.L.; Verkleij, A. J.; Kruijff, B. de

    1987-01-01

    The interaction of melittin with bovine heart cardiolipin model membranes was investigated via binding assays, 31P-NMR, freeze-fracture electron microscopy, small angle X-ray diffraction and fluorescence based fusion assays. A strong binding (Kd < 10−7 M) appeared to be accompanied by the formation of large structures, resulting from a fusion process of extremely fast initial rate. As the melittin content is increased, bilayer structure is gradually lost and from a cardiolipin to melittin rat...

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

    OpenAIRE

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

    2011-01-01

    The present study attempted to evaluate whether soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate lysosome fusion in response to death receptor activation and contribute to membrane raft (MR) clustering and consequent endothelial dysfunction in coronary arterial endothelial cells. By immunohistochemical analysis, vesicle-associated membrane proteins 2 (VAMP-2, vesicle-SNAREs) were found to be abundantly expressed in the endothelium of bovine coronary arte...

  10. Fusion of the Endoplasmic Reticulum and Mitochondrial Outer Membrane in Rats Brown Adipose Tissue: Activation of Thermogenesis by Ca2+

    OpenAIRE

    de Meis, Leopoldo; Ketzer, Luisa A.; da Costa, Rodrigo Madeiro; de Andrade, Ivone Rosa; Benchimol, Marlene

    2010-01-01

    Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca2+-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca2+ effect in BAT mitochondria thermogenesis. We found that Ca2+ increased the rate of respiration and heat production ...

  11. 硫醇-磷脂混合双层膜的电化学性质及其与蜂毒素的相互作用研究%Electrochemical Properties of Hybrid Bilayer Membranes and Interaction with Melittin

    Institute of Scientific and Technical Information of China (English)

    高宏; 罗国安; 冯军; Ottova,A.L.; Tien,H.T.

    2001-01-01

    报道了硫醇-磷脂混合双层膜的循环伏安和电化学交流阻抗行为研究,并用电化学方法考察了蜂毒素与其相互作用.实验中通过冷冻表面沾有磷脂溶液的硫醇单层膜来制备混合双层膜.研究表明双层膜在电极表面形成致密的绝缘层,阻碍了电极表面的电子传递.在双层膜体系上引入的蜂毒素可在膜表面上形成孔洞,破坏膜的绝缘性,降低膜电阻,增加膜电容,使带负电的探针Fe(CN)63-的氧化还原反应速度加快.%Hybrid bilayer membranes were fabricated on monolayer of alkanethiols self-assembled on gold electrode by freezing the monolayer coated with lipid solution for a few minutes. The resulted bilayer membranes were characterized with cyclic voltammetry and ac impedance. The interaction of a pore-forming peptide,melittin with such bilayer membranes was investigated with electrochemical techniques. The presence of hybrid bilayer membranes on electrode shows remarkable insulating properties by greatly attenuating the electron transfer between soluble redox couple. Adsorption of melittin into HBM destroys the insulating capability of HBM, causing membrane resistance to decrease and membrane capacitance to increase, thus accelerating the redox reaction for the negatively charged redox couple Fe(CN)63- .

  12. Cholesterol's location in lipid bilayers.

    Science.gov (United States)

    Marquardt, Drew; Kučerka, Norbert; Wassall, Stephen R; Harroun, Thad A; Katsaras, John

    2016-09-01

    It is well known that cholesterol modifies the physical properties of lipid bilayers. For example, the much studied liquid-ordered Lo phase contains rapidly diffusing lipids with their acyl chains in the all trans configuration, similar to gel phase bilayers. Moreover, the Lo phase is commonly associated with cholesterol-enriched lipid rafts, which are thought to serve as platforms for signaling proteins in the plasma membrane. Cholesterol's location in lipid bilayers has been studied extensively, and it has been shown - at least in some bilayers - to align differently from its canonical upright orientation, where its hydroxyl group is in the vicinity of the lipid-water interface. In this article we review recent works describing cholesterol's location in different model membrane systems with emphasis on results obtained from scattering, spectroscopic and molecular dynamics studies. PMID:27056099

  13. Class II fusion protein of alphaviruses drives membrane fusion through the same pathway as class I proteins

    OpenAIRE

    Zaitseva, Elena; Mittal, Aditya; Griffin, Diane E.; Chernomordik, Leonid V.

    2005-01-01

    Viral fusion proteins of classes I and II differ radically in their initial structures but refold toward similar conformations upon activation. Do fusion pathways mediated by alphavirus E1 and influenza virus hemagglutinin (HA) that exemplify classes II and I differ to reflect the difference in their initial conformations, or concur to reflect the similarity in the final conformations? Here, we dissected the pathway of low pH–triggered E1-mediated cell–cell fusion by reducing the numbers of a...

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

    Science.gov (United States)

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

    2010-11-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-07-01

    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

  17. Early Events in Chikungunya Virus Infection—From Virus CellBinding to Membrane Fusion

    Directory of Open Access Journals (Sweden)

    Mareike K. S. van Duijl-Richter

    2015-07-01

    Full Text Available 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.

  18. Reconstitution of Human Ion Channels into Solvent-free Lipid Bilayers Enhanced by Centrifugal Forces.

    Science.gov (United States)

    Hirano-Iwata, Ayumi; Ishinari, Yutaka; Yoshida, Miyu; Araki, Shun; Tadaki, Daisuke; Miyata, Ryusuke; Ishibashi, Kenichi; Yamamoto, Hideaki; Kimura, Yasuo; Niwano, Michio

    2016-05-24

    Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Nav1.5), and the human GABAA receptor (GABAAR) channel were formed, and the functional reconstitution of the channels into BLMs via vesicle fusion was investigated. Ion channel currents were recorded in 67% of the BLMs that were centrifuged with membrane vesicles under appropriate centrifugal conditions (14-55 × g). The characteristic channel properties were retained for hERG, Nav1.5, and GABAAR channels after centrifugal incorporation into the BLMs. A comparison of the centrifugal force with reported values for the fusion force revealed that a centrifugal enhancement in vesicle fusion was attained, not by accelerating the fusion process but by accelerating the delivery of membrane vesicles to the surface of the BLMs, which led to an increase in the number of membrane vesicles that were available for fusion. Our method for enhancing the probability of vesicle fusion promises to dramatically increase the experimental efficiency of BLM reconstitution systems, leading to the realization of a BLM-based, high-throughput platform for functional assays of various membrane proteins. PMID:27224486

  19. Effect of a glucose-triazole-hydrogenated cardanol conjugate on lipid bilayer membrane organization and thermotropic phase transition

    Science.gov (United States)

    Swain, Jitendriya; Kamalraj, M.; Surya Prakash Rao, H.; Mishra, Ashok K.

    2015-02-01

    This work focuses on the membrane perturbation, solubilisation and thermotropic phase transition process of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) MLVs induced by a glucose-triazole-hydrogenated cardanol conjugate (GTHCC). GTHCC is a recently introduced non toxic sugar derivative. Differential scanning calorimetry (DSC) and fluorescence molecular probe based techniques have been used to understand the concentration dependent membrane perturbation, solubilisation and thermotropic phase transition process of DPPC MLVs. The phase transition temperature of DPPC MLVs decreases with increase in mol% of GTHCC. At higher concentration above 10 mol%, GTHCC was significantly perturbed the membrane organization. The intrinsic fluorescence of GTHCC is also found to be sensitive towards phase behaviour and changes in membrane organization of DPPC MLVs.

  20. The role of membrane viscosity in the dynamics of fluid membranes

    CERN Document Server

    Arroyo, Marino; Heltai, Luca

    2010-01-01

    Fluid membranes made out of lipid bilayers are the fundamental separation structure in eukaryotic cells. Many physiological processes rely on dramatic shape and topological changes (e.g. fusion, fission) of fluid membrane systems. Fluidity is key to the versatility and constant reorganization of lipid bilayers. Here, we study the role of the membrane intrinsic viscosity, arising from the friction of the lipid molecules as they rearrange to accommodate shape changes, in the dynamics of morphological changes of fluid vesicles. In particular, we analyze the competition between the membrane viscosity and the viscosity of the bulk fluid surrounding the vesicle as the dominant dissipative mechanism. We consider the relaxation dynamics of fluid vesicles put in an out-of-equilibrium state, but conclusions can be drawn regarding the kinetics or power consumption in regulated shape changes in the cell. On the basis of numerical calculations, we find that the dynamics arising from the membrane viscosity are qualitativel...

  1. Insulin-stimulated Plasma Membrane Fusion of Glut4 Glucose Transporter-containing Vesicles Is Regulated by Phospholipase D1D⃞

    OpenAIRE

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

    2005-01-01

    Insulin stimulates glucose uptake in fat and muscle by mobilizing Glut4 glucose transporters from intracellular membrane storage sites to the plasma membrane. This process requires the trafficking of Glut4-containing vesicles toward the cell periphery, docking at exocytic sites, and plasma membrane fusion. We show here that phospholipase D (PLD) production of the lipid phosphatidic acid (PA) is a key event in the fusion process. PLD1 is found on Glut4-containing vesicles, is activated by insu...

  2. Line-Tension Controlled Mechanism for Influenza Fusion

    OpenAIRE

    Herre Jelger Risselada; Giovanni Marelli; Marc Fuhrmans; Smirnova, Yuliya G.; Helmut Grubmüller; Siewert Jan Marrink; Marcus Müller

    2012-01-01

    Our molecular simulations reveal that wild-type influenza fusion peptides are able to stabilize a highly fusogenic pre-fusion structure, i.e. a peptide bundle formed by four or more trans-membrane arranged fusion peptides. We rationalize that the lipid rim around such bundle has a non-vanishing rim energy (line-tension), which is essential to (i) stabilize the initial contact point between the fusing bilayers, i.e. the stalk, and (ii) drive its subsequent evolution. Such line-tension controll...

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

    Directory of Open Access Journals (Sweden)

    Kristen S Swithers

    Full Text Available 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.

  4. Convenient synthesis and application of versatile nucleic acid lipid membrane anchors in the assembly and fusion of liposomes

    DEFF Research Database (Denmark)

    Ries, Oliver; Löffler, Philipp M. G.; Vogel, Stefan

    2015-01-01

    Hydrophobic moieties like lipid membrane anchors are highly demanded modifications for nucleic acid oligomers. Membrane-anchor modified oligonucleotides are applicable in biomedicine leading to new delivery strategies as well as in biophysical investigations towards assembly and fusion of liposomes...... or the construction of DNA origami structures. We herein present the synthesis and applications of versatile lipid membrane anchor building blocks suitable for solid phase oligonucleotide synthesis. These are readily synthesized in bulk in five to seven steps from commercially available precursors...... and can be incorporated at any position within an oligonucleotide without significantly altering duplex stability and structure as proven by thermal denaturation experiments and circular dichroism. Furthermore, applicability could be demonstrated by assembly and fusion of liposomes mediated by lipid...

  5. Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion

    DEFF Research Database (Denmark)

    Søe, Kent; Andersen, Thomas Lykke; Hobolt-Pedersen, Anne-Sofie;

    2011-01-01

    mechanistic point of view, it is interesting that the distribution of syncytin-1 immunoreactivity on the cell surface parallels that of actin, another important player in cell fusion, and that cell-cell proximity induces particular patterns of distribution of syncytin-1 and actin in the respective cells......Generation of osteoclasts through fusion of mono-nucleated precursors is a key event of bone physiology and bone resorption is inefficient without osteoclast fusion. Several factors playing a critical role in the fusion process have already been recognized, but the factors involved in the actual...... fusion of the lipid bilayers of their cell membranes are still unknown. Syncytin-1 is a protein encoded by a human endogenous retroviral gene which was stably integrated into the human ancestor genome more than 24 million years ago. Upon activation, syncytin-1 is able to destabilize the lipid bilayer of...

  6. Lipid domain formation and ligand-receptor distribution in lipid bilayer membranes investigated by atomic force microscopy

    DEFF Research Database (Denmark)

    Kaasgaard, Thomas; Mouritsen, O.G.; Jørgensen, K.

    A novel experimental technique, based on atomic force microscopy (AFM), is proposed to visualize the lateral organization of membrane systems in the nanometer range. The technique involves the use of a ligand-receptor pair, biotin-avidin, which introduces a height variation on a solid-supported l......A novel experimental technique, based on atomic force microscopy (AFM), is proposed to visualize the lateral organization of membrane systems in the nanometer range. The technique involves the use of a ligand-receptor pair, biotin-avidin, which introduces a height variation on a solid...

  7. V-ATPase, ScNhxlp and Yeast Vacuole Fusion

    Institute of Scientific and Technical Information of China (English)

    Quan-Sheng Qiu

    2012-01-01

    Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos.It is a central cellular reaction that plays important roles in signal transduction,protein sorting and subcellular compartmentation.Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summanzed in this article.It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhxlp are key components of the vacuole fusion machinery in yeast.Yeast ScNhxlp regulates vacuole fusion by controlling the luminal pH.V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast.Fission defects are epistatic to fusion defects.Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast,the fusion reaction does not need the transport activity but requires the physical presence of the proton pump.Vo,the membrane-integral sector of the V-ATPase,forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the Vo trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.

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

    International Nuclear Information System (INIS)

    Research highlights: → Sifuvirtide interacts with erythrocyte and lymphocyte membrane in a concentration dependent manner by decreasing its dipole potential. → Dipole potential variations in lipid vesicles show sifuvirtide's lipid selectivity towards saturated phosphatidylcholines. → 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.

  9. Low pH-dependent Hepatitis C Virus Membrane Fusion Depends on E2 Integrity, Target Lipid Composition, and Density of Virus Particles*

    OpenAIRE

    Haid, Sibylle; Pietschmann, Thomas; Pécheur, Eve-Isabelle

    2009-01-01

    Hepatitis C virus (HCV) is an enveloped, positive strand RNA virus of about 9.6 kb. Like all enveloped viruses, the HCV membrane fuses with the host cell membrane during the entry process and thereby releases the genome into the cytoplasm, initiating the viral replication cycle. To investigate the features of HCV membrane fusion, we developed an in vitro fusion assay using cell culture-produced HCV and fluorescently labeled liposomes. With this model we could show that HCV-mediated fusion can...

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

    International Nuclear Information System (INIS)

    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 YxxΦ 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-1NL4.3 compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

  11. Biopolymer-Lipid Bilayer Interaction Modulates the Physical Properties of Liposomes: Mechanism and Structure.

    Science.gov (United States)

    Tan, Chen; Zhang, Yating; Abbas, Shabbar; Feng, Biao; Zhang, Xiaoming; Xia, Wenshui; Xia, Shuqin

    2015-08-19

    This study was conducted to elucidate the conformational dependence of the modulating ability of chitosan, a positively charged biopolymer, on a new type of liposome composed of mixed lipids including egg yolk phosphatidylcholine (EYPC) and nonionic surfactant (Tween 80). Analysis of the dynamic and structure of bilayer membrane upon interaction with chitosan by fluorescence and electron paramagnetic resonance techniques demonstrated that, in addition to providing a physical barrier for the membrane surface, the adsorption of chitosan extended and crimped chains rigidified the lipid membrane. However, the decrease in relative microviscosity and order parameter suggested that the presence of chitosan coils disturbed the membrane organization. It was also noted that the increase of fluidity in the lipid bilayer center was not pronounced, indicating the shallow penetration of coils into the hydrophobic interior of bilayer. Microscopic observations revealed that chitosan adsorption not only affected the morphology of liposomes but also modulated the particle aggregation and fusion. Especially, a number of very heterogeneous particles were visualized, which tended to confirm the role of chitosan coils as a "polymeric surfactant". In addition to particle deformation, the membrane permeability was also tuned. These findings may provide a new perspective to understand the physiological functionality of biopolymer and design biopolymer-liposome composite structures as delivery systems for bioactive components. PMID:26173584

  12. Fragmented state of lipid bilayers in water

    DEFF Research Database (Denmark)

    Helfrich, W.; Thimmel, J.; Klösgen, Beate Maria

    1999-01-01

    The bilayers of some typical biological membrane lipids such as PC and DGDG disintegrate in a large excess of water to form an optically invisible dispersive bilayer phase. `Dark bodies' can be reversibly precipitated from it by raising the temperature. The dispersive phase probably consists of...... `knotted sticks', i.e. very thin nodular tubes of bilayer. After reviewing pertinent experimental and theoretical work we report on the discovery of a lower consolute point near room temperature in DGDG/water systems. Its existence shows that the dispersive phase and the dark bodies belong to the same...... fragmented (or nodular) bilayer state, representing its expanded and condensed phases, respectively, above the critical temperature....

  13. Hemi-fused structure mediates and controls fusion and fission in live cells.

    Science.gov (United States)

    Zhao, Wei-Dong; Hamid, Edaeni; Shin, Wonchul; Wen, Peter J; Krystofiak, Evan S; Villarreal, Seth A; Chiang, Hsueh-Cheng; Kachar, Bechara; Wu, Ling-Gang

    2016-06-23

    Membrane fusion and fission are vital for eukaryotic life. For three decades, it has been proposed that fusion is mediated by fusion between the proximal leaflets of two bilayers (hemi-fusion) to produce a hemi-fused structure, followed by fusion between the distal leaflets, whereas fission is via hemi-fission, which also produces a hemi-fused structure, followed by full fission. This hypothesis remained unsupported owing to the lack of observation of hemi-fusion or hemi-fission in live cells. A competing fusion hypothesis involving protein-lined pore formation has also been proposed. Here we report the observation of a hemi-fused Ω-shaped structure in live neuroendocrine chromaffin cells and pancreatic β-cells, visualized using confocal and super-resolution stimulated emission depletion microscopy. This structure is generated from fusion pore opening or closure (fission) at the plasma membrane. Unexpectedly, the transition to full fusion or fission is determined by competition between fusion and calcium/dynamin-dependent fission mechanisms, and is notably slow (seconds to tens of seconds) in a substantial fraction of the events. These results provide key missing evidence in support of the hemi-fusion and hemi-fission hypothesis in live cells, and reveal the hemi-fused intermediate as a key structure controlling fusion and fission, as fusion and fission mechanisms compete to determine the transition to fusion or fission. PMID:27309816

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. The influence of non polar and polar molecules in mouse motile cells membranes and pure lipid bilayers.

    Directory of Open Access Journals (Sweden)

    Francisco J Sierra-Valdez

    Full Text Available We report an experimental study of mouse sperm motility that shows chief aspects characteristic of neurons: the anesthetic (produced by tetracaine and excitatory (produced by either caffeine or calcium effects and their antagonic action. While tetracaine inhibits sperm motility and caffeine has an excitatory action, the combination of these two substances balance the effects, producing a motility quite similar to that of control cells. We also study the effects of these agents (anesthetic and excitatory on the melting points of pure lipid liposomes constituted by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC and dipalmitoyl phosphatidic acid (DPPA. Tetracaine induces a large fluidization of the membrane, shifting the liposomes melting transition temperature to much lower values. The effect of caffeine is null, but its addition to tetracaine-doped liposomes greatly screen the fluidization effect. A high calcium concentration stiffens pure lipid membranes and strongly reduces the effect of tetracaine. Molecular Dynamics Simulations are performed to further understand our experimental findings at the molecular level. We find a strong correlation between the effect of antagonic molecules that could explain how the mechanical properties suitable for normal cell functioning are affected and recovered.

  16. Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.

    OpenAIRE

    Vermeer, J.E.M.; Munster, van, B.C.; Vischer, N O; Gadella, Th.W.J.

    2004-01-01

    Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowp...

  17. ER-associated SNAREs and Sey1p mediate nuclear fusion at two distinct steps during yeast mating

    OpenAIRE

    Rogers, Jason V.; Arlow, Tim; Inkellis, Elizabeth R.; Koo, Timothy S.; Rose, Mark D.

    2013-01-01

    During yeast mating, two haploid nuclei fuse membranes to form a single diploid nucleus. However, the known proteins required for nuclear fusion are unlikely to function as direct fusogens (i.e., they are unlikely to directly catalyze lipid bilayer fusion) based on their predicted structure and localization. Therefore we screened known fusogens from vesicle trafficking (soluble N-ethylmaleimide–sensitive factor attachment protein receptors [SNAREs]) and homotypic endoplasmic reticulum (ER) fu...

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

    DEFF Research Database (Denmark)

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

    1995-01-01

    assessed by flotation on sucrose density gradients, was not dependent on the presence of fusion-competent or fusion-incompetent sphingolipids in the liposomes. The results of this study support the notion that a stereospecific interaction of the viral fusion protein with D-erythro sphingolipids in the...

  19. A hemi-fission intermediate links two mechanistically distinct stages of membrane fission

    OpenAIRE

    Mattila, Juha-Pekka; Shnyrova, Anna V.; Sundborger, Anna C.; Hortelano, Eva Rodriguez; Fuhrmans, Marc; Neumann, Sylvia; Müller, Marcus; Hinshaw, Jenny E.; Schmid, Sandra L; Frolov, Vadim A.

    2015-01-01

    Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate 1,2 , characterized by a ‘stalk’ in which only the inner monolayers of the two compartments have merged to form a localized non-bilayer connection 1-3 . Formation of the hemi-fission intermediate requires energy input from proteins catalyzing membrane remodeling; however the relationship between protein conformational rearrangements and hemi...

  20. Fusion of liposomes with the plasma membrane of epithelial cells: Fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections

    OpenAIRE

    Knoll, G.; Burger, K.N.J.; Bron, R.; van Meer, G.; Verkleij, A. J.

    1988-01-01

    The fusion of liposomes with the plasma membrane of influenza virus- infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processi...

  1. Fusion of liposomes with the plasma membrane of epithelial cells: Fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections

    OpenAIRE

    Knoll, G; Burger, K.N.J.; Bron, R.; van Meer, G.; Verkleij, A J

    1988-01-01

    The fusion of liposomes with the plasma membrane of influenza virus-infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry, 24: 3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processi...

  2. Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections

    OpenAIRE

    1988-01-01

    The fusion of liposomes with the plasma membrane of influenza virus- infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry. 24:3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apical and basolateral plasma membrane domains after fusion was assessed by autoradiography of liposomal [3H]dipalmitoylphosphatidylcholine after rapid freezing or chemical fixation and further processi...

  3. Bilayers merge even when exocytosis is transient

    OpenAIRE

    Taraska, Justin W; Almers, Wolfhard

    2004-01-01

    During exocytosis, the lumen of secretory vesicles connects with the extracellular space. In some vesicles, this connection closes again, causing the vesicle to be recaptured mostly intact. The degree to which the bilayers of such vesicles mix with the plasma membrane is unknown. Work supporting the kiss-and-run model of transient exocytosis implies that synaptic vesicles allow neither lipid nor protein to escape into the plasma membrane, suggesting that the two bilayers never merge. Here, we...

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

    International Nuclear Information System (INIS)

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

  5. Line-tension controlled mechanism for influenza fusion.

    Directory of Open Access Journals (Sweden)

    Herre Jelger Risselada

    Full Text Available Our molecular simulations reveal that wild-type influenza fusion peptides are able to stabilize a highly fusogenic pre-fusion structure, i.e. a peptide bundle formed by four or more trans-membrane arranged fusion peptides. We rationalize that the lipid rim around such bundle has a non-vanishing rim energy (line-tension, which is essential to (i stabilize the initial contact point between the fusing bilayers, i.e. the stalk, and (ii drive its subsequent evolution. Such line-tension controlled fusion event does not proceed along the hypothesized standard stalk-hemifusion pathway. In modeled influenza fusion, single point mutations in the influenza fusion peptide either completely inhibit fusion (mutants G1V and W14A or, intriguingly, specifically arrest fusion at a hemifusion state (mutant G1S. Our simulations demonstrate that, within a line-tension controlled fusion mechanism, these known point mutations either completely inhibit fusion by impairing the peptide's ability to stabilize the required peptide bundle (G1V and W14A or stabilize a persistent bundle that leads to a kinetically trapped hemifusion state (G1S. In addition, our results further suggest that the recently discovered leaky fusion mutant G13A, which is known to facilitate a pronounced leakage of the target membrane prior to lipid mixing, reduces the membrane integrity by forming a 'super' bundle. Our simulations offer a new interpretation for a number of experimentally observed features of the fusion reaction mediated by the prototypical fusion protein, influenza hemagglutinin, and might bring new insights into mechanisms of other viral fusion reactions.

  6. On the microscopic and mesoscopic perturbations of lipid bilayers upon interaction with the MPER domain of the HIV glycoprotein gp41.

    Science.gov (United States)

    Oliva, Rosario; Emendato, Alessandro; Vitiello, Giuseppe; De Santis, Augusta; Grimaldi, Manuela; D'Ursi, Anna Maria; Busi, Elena; Del Vecchio, Pompea; Petraccone, Luigi; D'Errico, Gerardino

    2016-08-01

    The effect of the 665-683 fragment of the HIV fusion glycoprotein 41, corresponding to the MPER domain of the protein and named gp41MPER, on the microscopic structure and mesoscopic arrangement of palmitoyl oleoyl phosphatidylcholine (POPC) and POPC/sphingomyelin (SM)/cholesterol (CHOL) lipid bilayers is analyzed. The microscopic structuring of the bilayers has been studied by Electron Spin Resonance (ESR) spectroscopy, using glycerophosphocholines spin-labelled in different positions along the acyl chain. Transitions of the bilayer liquid crystalline state have been also monitored by Differential Scanning Calorimetry (DSC). Changes of the bilayers morphology have been studied by determining the dimension of the liposomes through Dynamic Light Scattering (DLS) measurements. The results converge in showing that the sample preparation procedure, the bilayer composition and the peptide/lipid ratio critically tune the lipid response to the peptide/membrane interaction. When gp41MPER is added to preformed liposomes, it positions at the bilayer interface and the lipid perturbation is limited to the more external segments. In contrast, if the peptide is mixed with the lipids during the liposome preparation, it assumes a trans-membrane topology. This happens at all peptide/lipid ratios for fluid POPC bilayers, while in the case of rigid POPC/SM/CHOL membranes a minimum ratio has to be reached, thus suggesting peptide self-aggregation to occur. Peptide insertion results in a dramatic increase of the lipid ordering and bilayer stiffening, which reflect in significant changes in liposome average dimension and distribution. The biological implications of these findings are discussed. PMID:27179640

  7. Chloroquine Increases Glucose Uptake via Enhancing GLUT4 Translocation and Fusion with the Plasma Membrane in L6 Cells

    Directory of Open Access Journals (Sweden)

    Qi Zhou

    2016-05-01

    Full Text Available Background/Aims: Chloroquine can induce an increase in the cellular uptake of glucose; however, the underlying mechanism is unclear. Methods: In this study, translocation of GLUT4 and intracellular Ca2+ changes were simultaneously observed by confocal microscope in L6 cells stably over-expressing IRAP-mOrange. The GLUT4 fusion with the plasma membrane (PM was traced using HA-GLUT4-GFP. Glucose uptake was measured using a cell-based glucose uptake assay. GLUT4 protein was detected by Western blotting and mRNA level was detected by RT-PCR. Results: We found that chloroquine induced significant increases in glucose uptake, glucose transporter GLUT4 translocation to the plasma membrane (GTPM, GLUT4 fusion with the PM, and intracellular Ca2+ in L6 muscle cells. Chloroquine-induced increases of GTPM and intracellular Ca2+ were inhibited by Gallein (Gβγ inhibitor and U73122 (PLC inhibitor. However, 2-APB (IP3R blocker only blocked the increase in intracellular Ca2+ but did not inhibit GTPM increase. These results indicate that chloroquine, via the Gβγ-PLC-IP3-IP3R pathway, induces elevation of Ca2+, and this Ca2+ increase does not play a role in chloroqui-ne-evoked GTPM increase. However, GLUT4 fusion with the PM and glucose uptake were significantly inhibited with BAPTA-AM. This suggests that Ca2+ enhances GLUT4 fusion with the PM resulting in glucose uptake increase. Conclusion: Our data indicate that chloroquine via Gβγ-PLC-IP3-IP3R induces Ca2+ elevation, which in turn promotes GLUT4 fusion with the PM. Moreover, chloroquine can enhance GLUT4 trafficking to the PM. These mechanisms eventually result in glucose uptake increase in control and insulin-resistant L6 cells. These findings suggest that chloroquine might be a potential drug for improving insulin tolerance in diabetic patients.

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

    International Nuclear Information System (INIS)

    Highlights: ► Initial conformational change of paramyxovirus F protein is caused only by mechanical forces. ► HRA region undergoes a structural change from a beta + alpha conformation to an extended coil and then to an all-alpha conformation. ► HRS domains of F protein form three single α-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 β-sheet conformation to an elongated coil and then spontaneously to an α-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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Cornelis, P.; Sierra, J.C.; Lim, A. Jr.; Malur, A. [Vrije Universiteit Brussel, Paardenstraat (Belgium)] [and others

    1996-02-01

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

  10. Structural criteria for regulation of membrane fusion and virion incorporation by the murine leukemia virus TM cytoplasmic domain

    International Nuclear Information System (INIS)

    The cytoplasmic domains of viral glycoproteins influence the trafficking and subcellular localization of the glycoproteins and their incorporation into virions. They also promote correct virus morphology and viral budding. The cytoplasmic domains of murine-leukemia-virus envelope-protein TM subunits regulate membrane fusion. During virion maturation the carboxy-terminal 16 amino acid residues of the TM protein are removed by the retroviral protease. Deletion of these residues activates envelope-protein-mediated membrane fusion. Our quantitative analysis of the effects of Moloney murine leukemia virus TM mutations on envelope-protein function support the proposition that a trimeric coiled coil in the TM cytoplasmic domain inhibits fusion. The data demonstrate that cleavage of the TM cytoplasmic domain is not required for viral entry and provide evidence for a model in which fusogenic and nonfusogenic conformations of the envelope protein exists in an equilibrium that is regulated by the cytoplasmic domain. In addition, a conserved tyrosine residue in the TM cytoplasmic domain was shown to play an important role in envelope-protein incorporation into retroviral particles

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

    Energy Technology Data Exchange (ETDEWEB)

    Martin-Garcia, Fernando; Mendieta-Moreno, Jesus Ignacio; Mendieta, Jesus [Centro de Biologia Molecular ' Severo Ochoa' (CSIC/UAM), C/ Nicolas Cabrera, 1, Cantoblanco, 28049 Madrid (Spain); Biomol-Informatics SL, Parque Cientifico de Madrid, C/ Faraday, 7, Cantoblanco, 28049 Madrid (Spain); Gomez-Puertas, Paulino, E-mail: pagomez@cbm.uam.es [Centro de Biologia Molecular ' Severo Ochoa' (CSIC/UAM), C/ Nicolas Cabrera, 1, Cantoblanco, 28049 Madrid (Spain)

    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.

  12. Fluid lipid bilayers: Intermonolayer coupling and its thermodynamic manifestations

    DEFF Research Database (Denmark)

    Hansen, Per Lyngs; Miao, Ling; Ipsen, John Hjorth

    1998-01-01

    A fluid membrane of lipid bilayer consists of two individual molecular monolayers physically opposed to each other. This unique molecular architecture naturally necessitates the need to treat a lipid-bilayer membrane as one entity of two coupled two-dimensional systems (monolayers), each of which...

  13. Development of high-performance ion sensors based on the functions of crown ethers and synthetic bilayer membranes; Kurauneteru oyobi goseinibunshimaku no kino wo kiban to shita koseino ion denkyoku no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)

    Yanagi, H.; Sakai, T.; Ogata, T. [Tokuyama Corp., Tokyo (Japan)

    1999-10-10

    High-Performance Na{sup +}, K{sup +} and Cl{sup -} ion selective electrodes (ISEs) are reported. These are based on a new concept, which is an ion recognition site formed by molecular interaction. In the case of Na and K ISEs, crown ether compounds connecting with planar substituents were used as the ionophores. The selectivities depend on the structure of substituents. It is found that the intermolecular interaction of the planar substituent plays an important role on the improvement of selectivity. The selectivities of interactive mono (crown ether) are equal to that of bis (crown ether). It means that ion recognition site like a bis (crown ether) is formed by the interaction of mono (crown ehter)s. In the case of C1 ISE, immobilized synthetic bilayer membrane with quaternary ammonium salts was used as the ion sensitive membrane. in the immobilized synthetic bilayer membrane, ordered molecular aggregates is formed. It is found that the molecular orientation improved the Cl selectivity. The insufficient selectivity over lipophilic anions can be addition of alphatic alcohol. These resulting ISEs exhibit high speed response and long life time, and they are applicable to clinical use which demands both accurate measurement and high speed processing. (author)

  14. Bilayer Effects of Antimalarial Compounds.

    Directory of Open Access Journals (Sweden)

    Nicole B Ramsey

    Full Text Available Because of the perpetual development of resistance to current therapies for malaria, the Medicines for Malaria Venture developed the Malaria Box to facilitate the drug development process. We tested the 80 most potent compounds from the box for bilayer-mediated effects on membrane protein conformational changes (a measure of likely toxicity in a gramicidin-based stopped flow fluorescence assay. Among the Malaria Box compounds tested, four compounds altered membrane properties (p< 0.05; MMV007384 stood out as a potent bilayer-perturbing compound that is toxic in many cell-based assays, suggesting that testing for membrane perturbation could help identify toxic compounds. In any case, MMV007384 should be approached with caution, if at all.

  15. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    International Nuclear Information System (INIS)

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

  16. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Zambetti, G.; Stein, J.; Stein, G.

    1987-05-01

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 ..beta..-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression.

  17. Effects of Concentrations on the Transdermal Permeation Enhancing Mechanisms of Borneol: A Coarse-Grained Molecular Dynamics Simulation on Mixed-Bilayer Membranes

    Science.gov (United States)

    Dai, Xingxing; Yin, Qianqian; Wan, Guang; Wang, Ran; Shi, Xinyuan; Qiao, Yanjiang

    2016-01-01

    Borneol is a natural permeation enhancer that is effective in drugs used in traditional clinical practices as well as in modern scientific research. However, its molecular mechanism is not fully understood. In this study, a mixed coarse-grained model of stratum corneum (SC) lipid bilayer comprised of Ceramide-N-sphingosine (CER NS) 24:0, cholesterol (CHOL) and free fatty acids (FFA) 24:0 (2:2:1) was used to examine the permeation enhancing mechanism of borneol on the model drug osthole. We found two different mechanisms that were dependent on concentrations levels of borneol. At low concentrations, the lipid system maintained a bilayer structure. The addition of borneol made the lipid bilayer loosen and improved drug permeation. The “pull” effect of borneol also improved drug permeation. However, for a strongly hydrophobic drug like osthole, the permeation enhancement of borneol was limited. When most borneol molecules permeated into bilayers and were located at the hydrophobic tail region, the spatial competition effect inhibited drug molecules from permeating deeper into the bilayer. At high concentrations, borneol led to the formation of water pores and long-lived reversed micelles. This improved the permeation of osthole and possibly other hydrophobic or hydrophilic drugs through the SC. Our simulation results were supported by Franz diffusion tests and transmission electron microscope (TEM) experiments. PMID:27548141

  18. Effects of Concentrations on the Transdermal Permeation Enhancing Mechanisms of Borneol: A Coarse-Grained Molecular Dynamics Simulation on Mixed-Bilayer Membranes

    Directory of Open Access Journals (Sweden)

    Xingxing Dai

    2016-08-01

    Full Text Available Borneol is a natural permeation enhancer that is effective in drugs used in traditional clinical practices as well as in modern scientific research. However, its molecular mechanism is not fully understood. In this study, a mixed coarse-grained model of stratum corneum (SC lipid bilayer comprised of Ceramide-N-sphingosine (CER NS 24:0, cholesterol (CHOL and free fatty acids (FFA 24:0 (2:2:1 was used to examine the permeation enhancing mechanism of borneol on the model drug osthole. We found two different mechanisms that were dependent on concentrations levels of borneol. At low concentrations, the lipid system maintained a bilayer structure. The addition of borneol made the lipid bilayer loosen and improved drug permeation. The “pull” effect of borneol also improved drug permeation. However, for a strongly hydrophobic drug like osthole, the permeation enhancement of borneol was limited. When most borneol molecules permeated into bilayers and were located at the hydrophobic tail region, the spatial competition effect inhibited drug molecules from permeating deeper into the bilayer. At high concentrations, borneol led to the formation of water pores and long-lived reversed micelles. This improved the permeation of osthole and possibly other hydrophobic or hydrophilic drugs through the SC. Our simulation results were supported by Franz diffusion tests and transmission electron microscope (TEM experiments.

  19. Effects of Concentrations on the Transdermal Permeation Enhancing Mechanisms of Borneol: A Coarse-Grained Molecular Dynamics Simulation on Mixed-Bilayer Membranes.

    Science.gov (United States)

    Dai, Xingxing; Yin, Qianqian; Wan, Guang; Wang, Ran; Shi, Xinyuan; Qiao, Yanjiang

    2016-01-01

    Borneol is a natural permeation enhancer that is effective in drugs used in traditional clinical practices as well as in modern scientific research. However, its molecular mechanism is not fully understood. In this study, a mixed coarse-grained model of stratum corneum (SC) lipid bilayer comprised of Ceramide-N-sphingosine (CER NS) 24:0, cholesterol (CHOL) and free fatty acids (FFA) 24:0 (2:2:1) was used to examine the permeation enhancing mechanism of borneol on the model drug osthole. We found two different mechanisms that were dependent on concentrations levels of borneol. At low concentrations, the lipid system maintained a bilayer structure. The addition of borneol made the lipid bilayer loosen and improved drug permeation. The "pull" effect of borneol also improved drug permeation. However, for a strongly hydrophobic drug like osthole, the permeation enhancement of borneol was limited. When most borneol molecules permeated into bilayers and were located at the hydrophobic tail region, the spatial competition effect inhibited drug molecules from permeating deeper into the bilayer. At high concentrations, borneol led to the formation of water pores and long-lived reversed micelles. This improved the permeation of osthole and possibly other hydrophobic or hydrophilic drugs through the SC. Our simulation results were supported by Franz diffusion tests and transmission electron microscope (TEM) experiments. PMID:27548141

  20. Virosome engineering of colloidal particles and surfaces: bioinspired fusion to supported lipid layers

    Science.gov (United States)

    Fleddermann, J.; Diamanti, E.; Azinas, S.; Košutić, M.; Dähne, L.; Estrela-Lopis, I.; Amacker, M.; Donath, E.; Moya, S. E.

    2016-04-01

    Immunostimulating reconstituted influenza virosomes (IRIVs) are liposomes with functional viral envelope glycoproteins: influenza virus hemagglutinin (HA) and neuraminidase intercalated in the phospholipid bilayer. Here we address the fusion of IRIVs to artificial supported lipid membranes assembled on polyelectrolyte multilayers on both colloidal particles and planar substrates. The R18 assay is used to prove the IRIV fusion in dependence of pH, temperature and HA concentration. IRIVs display a pH-dependent fusion mechanism, fusing at low pH in analogy to the influenza virus. The pH dependence is confirmed by the Quartz Crystal Microbalance technique. Atomic Force Microscopy imaging shows that at low pH virosomes are integrated in the supported membrane displaying flattened features and a reduced vertical thickness. Virosome fusion offers a new strategy for transferring biological functions on artificial supported membranes with potential applications in targeted delivery and sensing.Immunostimulating reconstituted influenza virosomes (IRIVs) are liposomes with functional viral envelope glycoproteins: influenza virus hemagglutinin (HA) and neuraminidase intercalated in the phospholipid bilayer. Here we address the fusion of IRIVs to artificial supported lipid membranes assembled on polyelectrolyte multilayers on both colloidal particles and planar substrates. The R18 assay is used to prove the IRIV fusion in dependence of pH, temperature and HA concentration. IRIVs display a pH-dependent fusion mechanism, fusing at low pH in analogy to the influenza virus. The pH dependence is confirmed by the Quartz Crystal Microbalance technique. Atomic Force Microscopy imaging shows that at low pH virosomes are integrated in the supported membrane displaying flattened features and a reduced vertical thickness. Virosome fusion offers a new strategy for transferring biological functions on artificial supported membranes with potential applications in targeted delivery and sensing

  1. Texture of lipid bilayer domains

    DEFF Research Database (Denmark)

    Jensen, Uffe Bernchou; Brewer, Jonathan R.; Midtiby, Henrik Skov; Ipsen, John Hjort; Bagatolli, Luis; Simonsen, Adam Cohen

    2009-01-01

    We investigate the texture of gel (g) domains in binary lipid membranes composed of the phospholipids DPPC and DOPC. Lateral organization of lipid bilayer membranes is a topic of fundamental and biological importance. Whereas questions related to size and composition of fluid membrane domain are...... chains. By imaging the intensity variations as a function of the polarization angle, we map the lateral variations of the lipid tilt within domains. Results reveal that gel domains are composed of subdomains with different lipid tilt directions. We have applied a Fourier decomposition method as a...... which correlates with the phase state of the membrane. This is quantified by the generalized polarization (GP) function, and we demonstrate that a GP analysis can be performed on supported membranes. The results show that although the gel domains have heterogeneous texture, the membrane phase state does...

  2. Drainin required for membrane fusion of the contractile vacuole in Dictyostelium is the prototype of a protein family also represented in man.

    Science.gov (United States)

    Becker, M; Matzner, M; Gerisch, G

    1999-01-01

    The contractile vacuole expels water by forming a channel with the plasma membrane and thus enables cells to survive in a hypo-osmotic environment. Here we characterize drainin, a Dictyostelium protein involved in this process, as the first member of a protein family represented in fission yeast, Caenorhabditis elegans and man. Gene replacement in Dictyostelium shows that drainin acts at a checkpoint of channel formation between the contractile vacuole and the plasma membrane. A green fluorescent protein fusion of drainin localizes specifically to the contractile vacuole and rescues its periodic discharge in drainin-null cells. Drainin is a peripheral membrane protein, requiring a short hydrophobic stretch in its C-terminal region for localization and function. We suggest that drainin acts in a signaling cascade that couples a volume-sensing device in the vacuolar membrane to the membrane fusion machinery. PMID:10369671

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

    International Nuclear Information System (INIS)

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

  4. Hydration effects on membrane structure probed by single molecule orientations.

    Science.gov (United States)

    Huckabay, Heath A; Dunn, Robert C

    2011-03-15

    Single molecule fluorescence measurements are used to probe the structural changes in glass-supported DPPC bilayers as a function of relative humidity (RH). Defocused polarized total internal reflection fluorescence microscopy is employed to determine the three-dimensional orientation of the fluorescent lipid analogue BODIPY-PC, doped into DPPC membranes in trace amounts. Supported DPPC bilayers formed using vesicle fusion and Langmuir-Blodgett/Langmuir-Schäfer (LB/LS) transfer are compared and show similar trends as a function of relative humidity. Population histograms of the emission dipole tilt angle reveal bimodal distributions as observed previously for BODIPY-PC in DPPC. These distributions are dominated by large populations of BODIPY-PC molecules with emission dipoles oriented parallel (≥81°) and normal (≤10°) to the membrane plane, with less than 25% oriented at intermediate tilts. As the relative humidity is increased from 13% to 95%, the population of molecules oriented normal to the surface decreases with a concomitant increase in those oriented parallel to the surface. The close agreement in trends observed for bilayers formed from vesicle fusion and LB/LS transfer supports the assignment of an equivalent surface pressure of 23 mN/m for bilayers formed from vesicle fusion. At each RH condition, a small population of BODIPY-PC dye molecules are laterally mobile in both bilayer preparations. This population exponentially increases with RH but never exceeds 6% of the total population. Interestingly, even under conditions where there is little lateral diffusion, fluctuations in the single molecule orientations can be observed which suggests there is appreciable freedom in the acyl chain region. Dynamic measurements of single molecule orientation changes, therefore, provide a new view into membrane properties at the single molecule level. PMID:21319764

  5. Biological and synthetic membranes: What can be learned from a coarse-grained description?

    International Nuclear Information System (INIS)

    We discuss the role coarse-grained models play in the investigation of the structure and thermodynamics of bilayer membranes, and we place them in the context of alternative approaches. Because they reduce the degrees of freedom and employ simple and soft effective potentials, coarse-grained models can provide rather direct insight into collective phenomena in membranes on large time and length scales. We present a summary of recent progress in this rapidly evolving field, and pay special attention to model development and computational techniques. Applications of coarse-grained models to changes of the membrane topology are illustrated with studies of membrane fusion utilizing simulations and self-consistent field theory

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

    Science.gov (United States)

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

    2016-08-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  8. DNA Duplexes with Hydrophobic Modifications Inhibit Fusion between HIV-1 and Cell Membranes

    OpenAIRE

    Xu, Liang; Cai, Lifeng; Chen, Xueliang; Jiang, Xifeng; Chong, Huihui; Zheng, Baohua; Wang, Kun; He, Junlin; Chen, Wei; Zhang, Tao; Cheng, Maosheng; He, Yuxian; Liu, Keliang

    2013-01-01

    Discovery of new drugs for the treatment of AIDS typically possessing unique structures associated with novel mechanisms of action has been of great importance due to the quick drug-resistant mutations of HIV-1 strains. The work presented in this report describes a novel class of DNA duplex-based HIV-1 fusion inhibitors. Hydrophobic groups were introduced into a DNA duplex skeleton either at one end, at both ends, or in the middle. These modified DNA duplexes inhibited fusion between HIV-1 an...

  9. Real-Time Structural Investigation of a Lipid Bilayer during Its Interaction with Melittin Using Sum Frequency Generation Vibrational Spectroscopy

    OpenAIRE

    Chen, Xiaoyun; Wang, Jie; Kristalyn, Cornelius B.; Chen, Zhan

    2007-01-01

    Interactions between membrane bilayers and peptides/proteins are ubiquitous throughout a cell. To determine the structure of membrane bilayers and the associated peptides/proteins, model systems such as supported lipid bilayers are often used. It has been difficult to directly investigate the interactions between a single membrane bilayer and peptides/proteins without exogenous labeling. In this work we demonstrate that sum frequency generation vibrational spectroscopy can be employed to stud...

  10. Fluidity evaluation of cell membrane model formed on graphene oxide with single particle tracking using quantum dot

    Science.gov (United States)

    Okamoto, Yoshiaki; Motegi, Toshinori; Iwasa, Seiji; Sandhu, Adarsh; Tero, Ryugo

    2015-04-01

    The lipid bilayer is the fundamental structure of plasma membranes, and artificial lipid bilayer membranes are used as model systems of cell membranes. Recently we reported the formation of a supported lipid bilayer (SLB) on graphene oxide (GO) by the vesicle fusion method. In this study, we conjugated a quantum dot (Qdot) on the SLB surface as a fluorescence probe brighter than dye-labeled lipid molecules, to qualitatively evaluate the fluidity of the SLB on GO by the single particle tracking method. We obtained the diffusion coefficient of the Qdot-conjugated lipids in the SLB on GO. We also performed the Qdot conjugation on the SLB containing a lipid conjugated with polyethylene glycol, to prevent the nonspecific adsorption of Qdots. The difference in the diffusion coefficients between the SLBs on the GO and the bare SiO2 regions was evaluated from the trajectory of single Qdot-conjugated lipid diffusing between the two regions.

  11. Bursting Bubbles and Bilayers

    Directory of Open Access Journals (Sweden)

    Steven P. Wrenn, Stephen M. Dicker, Eleanor F. Small, Nily R. Dan, Michał Mleczko, Georg Schmitz, Peter A. Lewin

    2012-01-01

    Full Text Available This paper discusses various interactions between ultrasound, phospholipid monolayer-coated gas bubbles, phospholipid bilayer vesicles, and cells. The paper begins with a review of microbubble physics models, developed to describe microbubble dynamic behavior in the presence of ultrasound, and follows this with a discussion of how such models can be used to predict inertial cavitation profiles. Predicted sensitivities of inertial cavitation to changes in the values of membrane properties, including surface tension, surface dilatational viscosity, and area expansion modulus, indicate that area expansion modulus exerts the greatest relative influence on inertial cavitation. Accordingly, the theoretical dependence of area expansion modulus on chemical composition - in particular, poly (ethylene glyclol (PEG - is reviewed, and predictions of inertial cavitation for different PEG molecular weights and compositions are compared with experiment. Noteworthy is the predicted dependence, or lack thereof, of inertial cavitation on PEG molecular weight and mole fraction. Specifically, inertial cavitation is predicted to be independent of PEG molecular weight and mole fraction in the so-called mushroom regime. In the “brush” regime, however, inertial cavitation is predicted to increase with PEG mole fraction but to decrease (to the inverse 3/5 power with PEG molecular weight. While excellent agreement between experiment and theory can be achieved, it is shown that the calculated inertial cavitation profiles depend strongly on the criterion used to predict inertial cavitation. This is followed by a discussion of nesting microbubbles inside the aqueous core of microcapsules and how this significantly increases the inertial cavitation threshold. Nesting thus offers a means for avoiding unwanted inertial cavitation and cell death during imaging and other applications such as sonoporation. A review of putative sonoporation mechanisms is then presented

  12. Direct in situ measurement of specific capacitance, monolayer tension, and bilayer tension in a droplet interface bilayer.

    Science.gov (United States)

    Taylor, Graham J; Venkatesan, Guru A; Collier, C Patrick; Sarles, Stephen A

    2015-10-14

    Thickness and tension are important physical parameters of model cell membranes. However, traditional methods to measure these quantities require multiple experiments using separate equipment. This work introduces a new multi-step procedure for directly accessing in situ multiple physical properties of droplet interface bilayers (DIB), including specific capacitance (related to thickness), lipid monolayer tension in the Plateau-Gibbs border, and bilayer tension. The procedure employs a combination of mechanical manipulation of bilayer area followed by electrowetting of the capacitive interface to examine the sensitivities of bilayer capacitance to area and contact angle to voltage, respectively. These data allow for determining the specific capacitance of the membrane and surface tension of the lipid monolayer, which are then used to compute bilayer thickness and tension, respectively. The use of DIBs affords accurate optical imaging of the connected droplets in addition to electrical measurements of bilayer capacitance, and it allows for reversibly varying bilayer area. After validating the accuracy of the technique with diphytanoyl phosphatidylcholine (DPhPC) DIBs in hexadecane, the method is applied herein to quantify separately the effects on membrane thickness and tension caused by varying the solvent in which the DIB is formed and introducing cholesterol into the bilayer. Because the technique relies only on capacitance measurements and optical images to determine both thickness and tension, this approach is specifically well-suited for studying the effects of peptides, biomolecules, natural and synthetic nanoparticles, and other species that accumulate within membranes without altering bilayer conductance. PMID:26289743

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

    Science.gov (United States)

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

    2016-07-01

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

  14. Role of lipid phase separations and membrane hydration in phospholipid vesicle fusion.

    NARCIS (Netherlands)

    Hoekstra, D.

    1982-01-01

    The relationship between lipid phase separation and fusion of small unilamellar phosphatidylserine-containing vesicles was investigated. The kinetics of phase separation were monitored by following the increase of self-quenching of the fluorescent phospholipid analogue N-(7-nitro-2,1,3-benzoxadiazol

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

    Energy Technology Data Exchange (ETDEWEB)

    Nayak, Vinod; Dessau, Moshe; Kucera, Kaury; Anthony, Karen; Ledizet, Michel; Modis, Yorgo; (Yale); (L2 Diagnostics)

    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.

  16. iMembrane: homology-based membrane-insertion of proteins

    OpenAIRE

    Kelm, Sebastian; Shi, Jiye; Deane, Charlotte M

    2009-01-01

    Summary: iMembrane is a homology-based method, which predicts a membrane protein's position within a lipid bilayer. It projects the results of coarse-grained molecular dynamics simulations onto any membrane protein structure or sequence provided by the user. iMembrane is simple to use and is currently the only computational method allowing the rapid prediction of a membrane protein's lipid bilayer insertion. Bilayer insertion data are essential in the accurate structural modelling of membrane...

  17. Micropipette manipulation technique for the monitoring of pH-dependent membrane lysis as induced by the fusion peptide of influenza virus.

    OpenAIRE

    Soltesz, S A; Hammer, D A

    1995-01-01

    We have assembled a micropipette aspiration assay to measure membrane destabilization events in which large (20-30 microns diameter) unilamellar vesicles are manipulated and exposed to membrane destabilizing agents. Single events can be seen with a light microscope and are recorded using both a video camera and a photomultiplier tube. We have performed experiments with a wild-type fusion peptide from influenza virus (X31) and found that it induces pH-dependent, stochastic lysis of large unila...

  18. Lipid Bilayers: Clusters, Domains and Phases

    OpenAIRE

    Ackerman, David G.; Feigenson, Gerald W.

    2015-01-01

    In this chapter we discuss the complex mixing behavior of plasma membrane lipids. To do so, we first introduce the plasma membrane and membrane mixtures often used to model its complexity. We then discuss the nature of lipid phase behavior in bilayers and the distinction between these phases and other manifestations of nonrandom mixing found in one-phase mixtures, such as clusters, micelles, and microemulsions. Finally, we demonstrate the applicability of Gibbs phase diagrams to the study of ...

  19. Ionic strength and composition govern the elasticity of biological membranes. A study of model DMPC bilayers by force- and transmission IR spectroscopy.

    Science.gov (United States)

    Šegota, Suzana; Vojta, Danijela; Pletikapić, Galja; Baranović, Goran

    2015-02-01

    Infrared (IR) spectroscopy was used to quantify the ion mixture effect of seawater (SW), particularly the contribution of Mg(2+) and Ca(2+) as dominant divalent cations, on the thermotropic phase behaviour of 1,2-dimyristoyl-sn-glycero-3-posphocholine (DMPC) bilayers. The changed character of the main transition at 24 °C from sharp to gradual in films and the 1 °C shift of the main transition temperature in dispersions reflect the interactions of lipid headgroups with the ions in SW. Force spectroscopy was used to quantify the nanomechanical hardness of a DMPC supported lipid bilayer (SLB). Considering the electrostatic and ion binding equilibrium contributions while systematically probing the SLB in various salt solutions, we showed that ionic strength had a decisive influence on its nanomechanics. The mechanical hardness of DMPC SLBs in the liquid crystalline phase linearly increases with the increasing fraction of all ion-bound lipids in a series of monovalent salt solutions. It also linearly increases in the gel phase but almost three times faster (the corresponding slopes are 4.9 nN/100 mM and 13.32 nN/100 mM, respectively). We also showed that in the presence of divalent ions (Ca(2+) and Mg(2+)) the bilayer mechanical hardness was unproportionally increased, and that was accompanied with the decrease of Na(+) ion and increase of Cl(-) ion bound lipids. The underlying process is a cooperative and competitive ion binding in both the gel and the liquid crystalline phase. Bilayer hardness thus turned out to be very sensitive to ionic strength as well as to ionic composition of the surrounding medium. In particular, the indicated correlation helped us to emphasize the colligative properties of SW as a naturally occurring complex ion mixture. PMID:25447291

  20. Structure and distribution of the Bacillus thuringiensis Cry4Ba toxin in lipid membranes

    International Nuclear Information System (INIS)

    Bacillus thuringiensis Cry δ-endotoxins cause death of susceptible insect larvae by forming lytic pores in the midgut epithelial cell membranes. The 65 kDa trypsin activated Cry4Ba toxin was previously shown to be capable of permeabilizing liposomes and forming ionic channels in receptor-free planar lipid bilayers. Here, magnetic ACmode (MACmode) atomic force microscopy (AFM) was used to characterize the lateral distribution and the native molecular structure of the Cry4Ba toxin in the membrane. Liposome fusion and the Langmuir-Blodgett technique were employed for supported lipid bilayer preparations. The toxin preferentially inserted in a self-assembled structure, rather than as a single monomeric molecule. In addition, the spontaneous insertion into receptor-free lipid bilayers lead to formation of characteristic pore-like structures with four-fold symmetry, suggesting that tetramers are the preferred oligomerization state of this toxin

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

    Science.gov (United States)

    Gao, Kai; Zhang, Yong; Lou, Jizhong

    2016-05-13

    Enveloped virus, such as HIV-1, employs membrane fusion mechanism to invade into host cell. HIV-1 gp41 ectodomain uses six-helix bundle configuration to accomplish this process. Using molecular dynamic simulations, we confirmed the stability of this six-helix bundle by showing high occupancy of hydrogen bonds and hydrophobic interactions. Key residues and interactions important for the bundle integration were characterized by force-induced unfolding simulations of six-helix bundle, exhibiting the collapse order of these groups of interactions. Moreover, our results in some way concerted with a previous theory that the formation of coiled-coil choose a route which involved cooperative interactions between the N-terminal and C-terminal helix. PMID:27079239

  2. Fusion of liposomes with the plasma membrane of epithelial cells: Fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections

    NARCIS (Netherlands)

    Knoll, G.; Burger, K.N.J.; Bron, R.; van Meer, G.; Verkleij, A.J.

    1988-01-01

    The fusion of liposomes with the plasma membrane of influenza virus-infected monolayers of an epithelial cell line, Madin-Darby canine kidney cells (van Meer et al., 1985. Biochemistry, 24: 3593-3602), has been analyzed by morphological techniques. The distribution of liposomal lipids over the apica

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

    Science.gov (United States)

    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. PMID:23510282

  4. Identification and purification of a sperm surface protein with a potential role in sperm-egg membrane fusion.

    Science.gov (United States)

    Primakoff, P; Hyatt, H; Tredick-Kline, J

    1987-01-01

    Sperm-egg plasma membrane fusion during fertilization was studied using guinea pig gametes and mAbs to sperm surface antigens. The mAb, PH-30, strongly inhibited sperm-egg fusion in a concentration-dependent fashion. When zona-free eggs were inseminated with acrosome-reacted sperm preincubated in saturating (140 micrograms/ml) PH-30 mAb, the percent of eggs showing fusion was reduced 75%. The average number of sperm fused per egg was also reduced by 75%. In contrast a control mAb, PH-1, preincubated with sperm at 400 micrograms/ml, caused no inhibition. The PH-30 and PH-1 mAbs apparently recognize the same antigen but bind to two different determinants. Both mAbs immunoprecipitated the same two 125I-labeled polypeptides with Mr 60,000 (60 kD) and Mr 44,000 (44 kD). Boiling a detergent extract of sperm severely reduced the binding of PH-30 but had essentially no effect on the binding of PH-1, indicating that the two mAbs recognize different epitopes. Immunoelectron microscopy revealed that PH-30 mAb binding was restricted to the sperm posterior head surface and was absent from the equatorial region. The PH-30 and PH-1 mAbs did not bind to sperm from the testis, the caput, or the corpus epididymis. PH-30 mAb binding was first detectable on sperm from the proximal cauda epididymis, i.e., sperm at the developmental stage where fertilization competence appears. After purification by mAb affinity chromatography, the PH-30 protein retained antigenic activity, binding both the PH-30 and PH-1 mAbs. The purified protein showed two polypeptide bands of 60 and 44 kD on reducing SDS PAGE. The two polypeptides migrated further (to approximately 49 kD and approximately 33 kD) on nonreducing SDS PAGE, showing that they do not contain interchain disulfide bonds, but probably have intrachain disulfides. 44 kD appears not to be a proteolytic fragment of 60 kD because V8 protease digestion patterns did not reveal related peptide patterns from the 44- and 60-kD bands. In the absence of

  5. Fusion of the endoplasmic reticulum and mitochondrial outer membrane in rats brown adipose tissue: activation of thermogenesis by Ca2+.

    Science.gov (United States)

    de Meis, Leopoldo; Ketzer, Luisa A; da Costa, Rodrigo Madeiro; de Andrade, Ivone Rosa; Benchimol, Marlene

    2010-01-01

    Brown adipose tissue (BAT) mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1), GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER) membrane with the mitochondrial outer membrane of rats BAT. Ca(2+)-ATPase (SERCA 1) was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca(2+) effect in BAT mitochondria thermogenesis. We found that Ca(2+) increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca(2+) concentration needed for half-maximal activation varied between 0.08 and 0.11 microM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN. Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca(2+) strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca(2+) activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver. PMID:20209153

  6. Gramicidin-based fluorescence assay; for determining small molecules potential for modifying lipid bilayer properties

    NARCIS (Netherlands)

    Ingólfsson, Helgi I; Sanford, R Lea; Kapoor, Ruchi; Andersen, Olaf S

    2010-01-01

    Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions.

  7. Screening for small molecules' bilayer-modifying potential using a gramicidin-based fluorescence assay

    NARCIS (Netherlands)

    Ingólfsson, Helgi I; Andersen, Olaf S

    2010-01-01

    Many drugs and other small molecules used to modulate biological function are amphiphiles that adsorb at the bilayer/solution interface and thereby alter lipid bilayer properties. This is important because membrane proteins are energetically coupled to their host bilayer by hydrophobic interactions.

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

    OpenAIRE

    Swithers, Kristen S.; Fournier, Gregory P.; Anna G Green; 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, ...

  9. A minimal phycobilisome: fusion and chromophorylation of the truncated core-membrane linker and phycocyanin.

    Science.gov (United States)

    Tang, Kun; Zeng, Xiao-Li; Yang, Yi; Wang, Zhi-Bin; Wu, Xian-Jun; Zhou, Ming; Noy, Dror; Scheer, Hugo; Zhao, Kai-Hong

    2012-07-01

    Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli, the fused phycobiliproteins absorbed light in the range of 610-660nm, and fluoresced at ~670nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540-575nm, and fluorescing at ~585nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly. PMID:22465853

  10. Formation of cholesterol-rich supported membranes using solvent-assisted lipid self-assembly.

    Science.gov (United States)

    Tabaei, Seyed R; Jackman, Joshua A; Kim, Seong-Oh; Liedberg, Bo; Knoll, Wolfgang; Parikh, Atul N; Cho, Nam-Joon

    2014-11-11

    This paper describes the application of a solvent-exchange method to prepare supported membranes containing high fractions of cholesterol (up to ∼57 mol %) in an apparent equilibrium. The method exploits the phenomenon of reverse-phase evaporation, in which the deposition of lipids in alcohol (e.g., isopropanol) is followed by the slow removal of the organic solvent from the water-alcohol mixture. This in turn induces a series of lyotropic phase transitions successively producing inverse-micelles, monomers, micelles, and vesicles in equilibrium with supported bilayers at the contacting solid surface. By using the standard cholesterol depletion by methyl-β-cyclodextrin treatment, a quartz crystal microbalance with dissipation monitoring assay confirms that the cholesterol concentration in the supported membranes is comparable to that in the surrounding bulk phase. A quantitative characterization of the biophysical properties of the resultant bilayer, including lateral diffusion constants and phase separation, using epifluorescence microscopy and atomic force microscopy establishes the formation of laterally contiguous supported lipid bilayers, which break into a characteristic domain-pattern of coexisting phases in a cholesterol concentration-dependent manner. With increasing cholesterol fraction in the supported bilayer, the size of the domains increases, ultimately yielding two-dimensional cholesterol bilayer domains near the solubility limit. A unique feature of the approach is that it enables preparation of supported membranes containing limiting concentrations of cholesterol near the solubility limit under equilibrium conditions, which cannot be obtained using conventional techniques (i.e., vesicle fusion). PMID:25286344

  11. Automatable lipid bilayer formation for ion channel studies

    Science.gov (United States)

    Poulos, Jason L.; Bang, Hyunwoo; Jeon, Tae-Joon; Schmidt, Jacob J.

    2008-08-01

    Transmembrane proteins and ion channels are important drug targets and have been explored as single molecule sensors. For these proteins to function normally they must be integrated within lipid bilayers; however, the labor and skill required to create artificial lipid bilayers have the limited the possible applications utilizing these proteins. In order to reduce the complexity and cost of lipid bilayer formation and measurement, we have modified a previously published lipid bilayer formation technique using mechanically contacted monolayers so that the process is automatable, requiring minimal operator input. Measurement electronics are integrated with the fluid handling system, greatly reducing the time and operator feedback characteristically required of traditional bilayer experiments. To demonstrate the biological functionality of the resultant bilayers and the system's capabilities as a membrane platform, the ion channel gramicidin A was incorporated and measured with this system.

  12. Dextran sulfate-dependent fusion of liposomes containing cationic stearylamine.

    Science.gov (United States)

    Zschörnig, O; Arnold, K; Richter, W; Ohki, S

    1992-11-01

    The incorporation of the positively charged stearylamine into phosphatidylcholine liposomes was studied by measuring electrophoretic mobilities. Up to a molar ratio SA/PC = 0.5 an increase of the positive zeta potential can be observed. Addition of the negatively charged macromolecule dextran sulfate leads to a change of the sign of the surface potential of the PC/SA liposomes indicating binding of the macromolecule to the surface. This process is accompanied by an increase in turbidity, which is dependent on the molecular weight of the dextran sulfate and the SA concentration (measured by turbidimetry). Using the NBD/Rh and Pyr-PC fluorescence assays the fusion of SA containing liposomes was investigated. A strong influence of the SA content and molecular weight of dextran sulfate on the fusion extent was observed. The fusion extent is proportional to the SA content in the PC membrane and the molecular weight of dextran sulfate. PC/SA/PE liposomes exhibit a higher fusion extent after addition of dextran sulfate compared to PC/SA liposomes indicating that PE additionally destabilizes the bilayer. Freeze-fracture electron microscopy reveals that the reaction products are large complexes composed of multilamellar stacks of tightly packed, straight membranes and aggregated vesicles. The tight packing of the membranes in the stacks (and the narrow contact of the aggregated vesicles) indicates a strong adherence of opposite membrane surfaces induced by dextran sulfate. PMID:1486657

  13. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  14. Characterization of lipid bilayer formation in aligned nanoporous aluminum oxide nanotube arrays.

    Science.gov (United States)

    Karp, Ethan S; Newstadt, Justin P; Chu, Shidong; Lorigan, Gary A

    2007-07-01

    Aligning lipid bilayers in nanoporous anodized aluminum oxide (AAO) is a new method to help study membrane proteins by electron paramagnetic resonance (EPR) and solid-state nuclear magnetic resonance (NMR) spectroscopic methods. The ability to maintain hydration, sample stability, and compartmentalization over long periods of time, and to easily change solvent composition are major advantages of this new method. To date, 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) has been the only phospholipid used for membrane protein studies with AAO substrates. The different properties of lipids with varying chain lengths require modified sample preparation procedures to achieve well formed bilayers within the lining of the AAO substrates. For the first time, the current study presents a simple methodology to incorporate large quantities of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), DMPC, and 1,2-dipalmitoyl-3-sn-phosphatidylcholine (DPPC) phospholipids inside AAO substrate nanopores of varying sizes. (2)H and (31)P solid-state NMR were used to confirm the alignment of each lipid and compare the efficiency of alignment. This study is the first step in standardizing the use of AAO substrates as a tool in NMR and EPR and will be useful for future structural studies of membrane proteins. Additionally, the solid-state NMR data suggest possible applications of nanoporous aluminum oxide in future vesicle fusion studies. PMID:17482492

  15. Implication des peptides de fusion des glycoprotéines de fusion virales de classe I dans la fusion membranaire

    OpenAIRE

    Brasseur R.; Charloteaux B.; Lins L.; Lorin A.

    2007-01-01

    The implication of fusion peptides of class I viral fusion glycoproteins in the membrane fusion. Viral infection involves fusion between the viral envelope and the target cell plasmic membrane. The fusion is induced by a glycoprotein anchored in the viral envelope. After activation, the glycoprotein undergoes a conformational change inducing the exposure of a region named « fusion peptide » essential for the fusion process. Studies on glycoproteins and on isolated fusion peptides have allowed...

  16. Superdiffusion in supported lipid bilayers

    CERN Document Server

    Campagnola, Grace; Schroder, Bryce W; Peersen, Olve B; Krapf, Diego

    2015-01-01

    We study the diffusion of membrane-targeting C2 domains using single-molecule tracking in supported lipid bilayers. The ensemble-averaged mean square displacement (MSD) exhibits superdiffusive behavior. However, the time-averaged MSD of individual trajectories is found to be linear with respect to lag time, as in Brownian diffusion. These observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution. Our experimental results are shown to agree with analytical models of bulk-mediated diffusion and with numerical simulations.

  17. Palmitoylation of SARS-CoV S protein is necessary for partitioning into detergent-resistant membranes and cell-cell fusion but not interaction with M protein

    International Nuclear Information System (INIS)

    Coronaviruses are enveloped RNA viruses that generally cause mild disease in humans. However, the recently emerged coronavirus that caused severe acute respiratory syndrome (SARS-CoV) is the most pathogenic human coronavirus discovered to date. The SARS-CoV spike (S) protein mediates virus entry by binding cellular receptors and inducing fusion between the viral envelope and the host cell membrane. Coronavirus S proteins are palmitoylated, which may affect function. Here, we created a non-palmitoylated SARS-CoV S protein by mutating all nine cytoplasmic cysteine residues. Palmitoylation of SARS-CoV S was required for partitioning into detergent-resistant membranes and for cell-cell fusion. Surprisingly, however, palmitoylation of S was not required for interaction with SARS-CoV M protein. This contrasts with the requirement for palmitoylation of mouse hepatitis virus S protein for interaction with M protein and may point to important differences in assembly and infectivity of these two coronaviruses.

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

    Science.gov (United States)

    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. PMID:26803470

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

    Energy Technology Data Exchange (ETDEWEB)

    Yasuzaki, Yukari; Yamada, Yuma [Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan); Harashima, Hideyoshi, E-mail: harasima@pharm.hokudai.ac.jp [Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan)

    2010-06-25

    Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we provide a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.

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

    International Nuclear Information System (INIS)

    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.

  1. Electrowetting on dielectric-based microfluidics for integrated lipid bilayer formation and measurement

    Science.gov (United States)

    Poulos, Jason L.; Nelson, Wyatt C.; Jeon, Tae-Joon; Kim, Chang-Jin ``Cj''; Schmidt, Jacob J.

    2009-07-01

    We present a microfluidic platform for the formation and electrical measurement of lipid bilayer membranes. Using electrowetting on dielectric (EWOD), two or more aqueous droplets surrounded by a lipid-containing organic phase were manipulated into contact to form a lipid bilayer at their interface. Thin-film Ag/AgCl electrodes integrated into the device enabled electrical measurement of membrane formation and the incorporation of gramicidin channels of two bilayers in parallel.

  2. The role of membrane fusion activity of a whole inactivated influenza virus vaccine in (re)activation of influenza-specific cytotoxic T lymphocytes.

    Science.gov (United States)

    Budimir, Natalija; Meijerhof, Tjarko; Wilschut, Jan; Huckriede, Anke; de Haan, Aalzen

    2010-12-01

    Induction of cytotoxic T lymphocyte (CTL) activity against conserved influenza antigens, e.g. nucleoprotein (NP) could be a step towards cross-protective influenza vaccine. The major challenge for non-replicating influenza vaccines aiming for activation of CTLs is targeting of antigen to the MHC class I processing and presentation pathway of professional antigen presenting cells, in particular dendritic cells (DCs). Intrinsic fusogenic properties of the vaccine particle itself can enable direct cytosolic delivery of the antigen by enhancing release of the antigen from the endosome to the cytosol. Alternatively, the vaccine particle would need to possess the capacity to activate DCs thereby triggering cell-intrinsic mechanisms of cross-presentation, processes that do not require fusion. Here, using fusion-active and fusion-inactive whole inactivated virus (WIV) as a vaccine model, we studied the relative contribution of these two pathways on priming and reactivation of influenza NP-specific CTLs in a murine model. We show that activation of bone marrow-derived DCs by WIV, as well as reactivation of NP-specific CTLs in vitro and in vivo were not affected by inactivation of membrane fusion of the WIV particles. However, in vivo priming of naive CTLs was optimal only upon vaccination with fusion-active WIV. Thus, DC-intrinsic mechanisms of cross-presentation are involved in the activation of CTLs upon vaccination with WIV. However, for optimal priming of naive CTLs these mechanisms should be complemented by delivery of antigen to the cytosol mediated by the membrane fusion capacity of the WIV particles. PMID:20965298

  3. SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

    International Nuclear Information System (INIS)

    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.

  4. Membrane Protein Structure Determination: Back to the Membrane

    OpenAIRE

    Yao, Yong; Ding, Yi; Tian, Ye; Opella, Stanley J.; Marassi, Francesca M.

    2013-01-01

    NMR spectroscopy enables the structures of membrane proteins to be determined in the native-like environment of the phospholipid bilayer membrane. This chapter outlines the methods for membrane protein structural studies using solid-state NMR spectroscopy with samples of membrane proteins incorporated in proteoliposomes or planar lipid bilayers. The methods for protein expression and purification, sample preparation, and NMR experiments are described and illustrated with examples from OmpX an...

  5. Amphiphile regulation of ion channel function by changes in the bilayer spring constant

    DEFF Research Database (Denmark)

    Lundbæk, Jens August; Koeppe, R.E.; Andersen, Oluf Sten

    2010-01-01

    predicted from measurements of isolated changes in such properties. Thus, the bilayer contribution to the promiscuous regulation of membrane proteins by drugs and other amphiphiles remains unknown. To overcome this problem, we use gramicidin A (gA) channels as molecular force probes to measure the net...... altering the energetic cost (Delta G(bilayer)) of bilayer deformations associated with protein conformational changes that involve the protein-bilayer interface. But amphiphiles have complex effects on the physical properties of lipid bilayers, meaning that the net change in Delta G(bilayer) cannot be......-dependent sodium channels in living cells. The use of gA channels as molecular force probes provides a tool for quantitative, predictive studies of bilayer-mediated regulation of membrane protein function by amphiphiles....

  6. Pushing the Envelope: Dengue Viral Membrane Coaxed into Shape by Molecular Simulations.

    Science.gov (United States)

    Marzinek, Jan K; Holdbrook, Daniel A; Huber, Roland G; Verma, Chandra; Bond, Peter J

    2016-08-01

    Dengue virus is a flavivirus responsible for millions of infections per year. Its surface contains a phospholipid bilayer, within which are embedded the envelope (E) and membrane (M) proteins, arranged with icosahedral geometry. Exposure to low pH triggers the E proteins to undergo conformational changes, which precede fusion with the host cell membrane and release of the viral genome. The flavivirus membrane exhibits significant local curvature and deformation, as revealed by cryoelectron microscopy (cryo-EM), but its precise structure and interactions with envelope components remain unclear. We now report simulations of the dengue viral particle that refine its envelope structure in unprecedented detail. Our final models are morphologically consistent with cryo-EM data, and reveal the structural basis for membrane curvature. Electrostatic interactions increased envelope complex stability; this coupling has potential functional significance in the context of the viral fusion mechanism and infective states. PMID:27396828

  7. Highly specific inhibition of leukaemia virus membrane fusion by interaction of peptide antagonists with a conserved region of the coiled coil of envelope

    Directory of Open Access Journals (Sweden)

    van Aalten Daan MF

    2008-08-01

    Full Text Available Abstract Background Human T-cell leukaemia virus (HTLV-1 and bovine leukaemia virus (BLV entry into cells is mediated by envelope glycoprotein catalyzed membrane fusion and is achieved by folding of the transmembrane glycoprotein (TM from a rod-like pre-hairpin intermediate to a trimer-of-hairpins. For HTLV-1 and for several virus groups this process is sensitive to inhibition by peptides that mimic the C-terminal α-helical region of the trimer-of-hairpins. Results We now show that amino acids that are conserved between BLV and HTLV-1 TM tend to map to the hydrophobic groove of the central triple-stranded coiled coil and to the leash and C-terminal α-helical region (LHR of the trimer-of-hairpins. Remarkably, despite this conservation, BLV envelope was profoundly resistant to inhibition by HTLV-1-derived LHR-mimetics. Conversely, a BLV LHR-mimetic peptide antagonized BLV envelope-mediated membrane fusion but failed to inhibit HTLV-1-induced fusion. Notably, conserved leucine residues are critical to the inhibitory activity of the BLV LHR-based peptides. Homology modeling indicated that hydrophobic residues in the BLV LHR likely make direct contact with a pocket at the membrane-proximal end of the core coiled-coil and disruption of these interactions severely impaired the activity of the BLV inhibitor. Finally, the structural predictions assisted the design of a more potent antagonist of BLV membrane fusion. Conclusion A conserved region of the HTLV-1 and BLV coiled coil is a target for peptide inhibitors of envelope-mediated membrane fusion and HTLV-1 entry. Nevertheless, the LHR-based inhibitors are highly specific to the virus from which the peptide was derived. We provide a model structure for the BLV LHR and coiled coil, which will facilitate comparative analysis of leukaemia virus TM function and may provide information of value in the development of improved, therapeutically relevant, antagonists of HTLV-1 entry into cells.

  8. Single DNA molecules on freestanding and supported cationic lipid bilayers: diverse conformational dynamics controlled by the local bilayer properties

    Science.gov (United States)

    Herold, Christoph; Schwille, Petra; Petrov, Eugene P.

    2016-02-01

    We present experimental results on the interaction of DNA macromolecules with cationic lipid membranes with different properties, including freestanding membranes in the fluid and gel state, and supported lipid membranes in the fluid state and under conditions of fluid-gel phase coexistence. We observe diverse conformational dynamics of membrane-bound DNA molecules controlled by the local properties of the lipid bilayer. In case of fluid-state freestanding lipid membranes, the behaviour of DNA on the membrane is controlled by the membrane charge density: whereas DNA bound to weakly charged membranes predominantly behaves as a 2D random coil, an increase in the membrane charge density leads to membrane-driven irreversible DNA collapse and formation of subresolution-sized DNA globules. On the other hand, electrostatic binding of DNA macromolecules to gel-state freestanding membranes leads to completely arrested diffusion and conformational dynamics of membrane-adsorbed DNA. A drastically different picture is observed in case of DNA interaction with supported cationic lipid bilayers: When the supported bilayer is in the fluid state, membrane-bound DNA molecules undergo 2D translational Brownian motion and conformational fluctuations, irrespectively of the charge density of the supported bilayer. At the same time, when the supported cationic membrane shows fluid-gel phase coexistence, membrane-bound DNA molecules are strongly attracted to micrometre-sized gel-phase domains enriched with the cationic lipid, which results in 2D compaction of the membrane-bound macromolecules. This DNA compaction, however, is fully reversible, and disappears as soon as the membrane is heated above the fluid-gel coexistence. We also discuss possible biological implications of our experimental findings.

  9. The importance of membrane defects-lessons from simulations.

    Science.gov (United States)

    Bennett, W F Drew; Tieleman, D Peter

    2014-08-19

    shape. The localization and trafficking of lipids supports general membrane structure and a number of important signaling cascades, such as those involving ceramide, diacylglycerol, and cholesterol. Atomistic simulations have determined the rates and free energies of lipid flip-flop. During the flip-flop of most phosphatidylcholine lipids, a hydrophilic pore forms when the headgroup moves near the center of the bilayer. Simulations have provided novel insight into many features of defects and pores in lipid membranes. Simulation data from very different systems and models show how water penetration and defect formation can determine the free energies of many membrane processes. Bilayers can deform and allow transient defects and pores when exposed to a diverse range of stimuli. Future work will explore many aspects of membrane defects with increased resolution and scope, including the study of more complex lipid mixtures, membrane domains, and large-scale membrane remodeling. Such studies will examine processes including vesicle budding and fusion, non-bilayer lipid phases, and interactions between lipid bilayers and other biomolecules. Simulations provide information that complements experimental studies, allowing microscopic insight into experimental observations and suggesting novel hypotheses and experiments. These studies should enable a deeper understanding of the role of lipid bilayers in cellular biology and support the development of future lipid-based biotechnology. PMID:24892900

  10. Flagellar membrane fusion and protein exchange in trypanosomes; a new form of cell-cell communication? [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    Simon Imhof

    2016-04-01

    Full Text Available 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.

  11. Molecular Dynamics of a Water-Lipid Bilayer Interface

    Science.gov (United States)

    Wilson, Michael A.; Pohorille, Andrew

    1994-01-01

    We present results of molecular dynamics simulations of a glycerol 1-monooleate bilayer in water. The total length of analyzed trajectories is 5ns. The calculated width of the bilayer agrees well with the experimentally measured value. The interior of the membrane is in a highly disordered fluid state. Atomic density profile, orientational and conformational distribution functions, and order parameters indicate that disorder increases toward the center of the bilayer. Analysis of out-of-plane thermal fluctuations of the bilayer surfaces occurring at the time scale of the present calculations reveals that the distribution of modes agrees with predictions of the capillary wave model. Fluctuations of both bilayer surfaces are uncorrelated, yielding Gaussian distribution of instantaneous widths of the membrane. Fluctuations of the width produce transient thinning defects in the bilayer which occasionally span almost half of the membrane. The leading mechanism of these fluctuations is the orientational and conformational motion of head groups rather than vertical motion of the whole molecules. Water considerably penetrates the head group region of the bilayer but not its hydrocarbon core. The total net excess dipole moment of the interfacial water points toward the aqueous phase, but the water polarization profile is non-monotonic. Both water and head groups significantly contribute to the surface potential across the interface. The calculated sign of the surface potential is in agreement with that from experimental measurements, but the value is markedly overestimated. The structural and electrical properties of the water-bilayer system are discussed in relation to membrane functions, in particular transport of ions and nonelectrolytes across membranes.

  12. CD81 and CD9 work independently as extracellular components upon fusion of sperm and oocyte

    Directory of Open Access Journals (Sweden)

    Naoko Ohnami

    2012-05-01

    When a sperm and oocyte unite into one cell upon fertilization, membranous fusion between the sperm and oocyte occurs. In mice, Izumo1 and a tetraspanin molecule CD9 are required for sperm-oocyte fusion as one of the oocyte factors, and another tetraspanin molecule CD81 is also thought to involve in this process. Since these two tetraspanins often form a complex upon cell-cell interaction, it is probable that such a complex is also formed in sperm-oocyte interaction; however, this possibility is still under debate among researchers. Here we assessed this problem using mouse oocytes. Immunocytochemical analysis demonstrated that both CD9 and CD81 were widely distributed outside the oocyte cell membrane, but these molecules were separate, forming bilayers, confirmed by immunobiochemical analysis. Electron-microscopic analysis revealed the presence of CD9- or CD81-incorporated extracellular structures in those bilayers. Finally, microinjection of in vitro-synthesized RNA showed that CD9 reversed a fusion defect in CD81-deficient oocytes in addition to CD9-deficient oocytes, but CD81 failed in both oocytes. These results suggest that both CD9 and CD81 independently work upon sperm-oocyte fusion as extracellular components.

  13. PMP1 18-38, a yeast plasma membrane protein fragment, binds phosphatidylserine from bilayer mixtures with phosphatidylcholine: a (2)H-NMR study.

    OpenAIRE

    M. Roux; Beswick, V; Coïc, Y M; Huynh-Dinh, T.; Sanson, A.; Neumann, J M

    2000-01-01

    PMP1 is a 38-residue plasma membrane protein of the yeast Saccharomyces cerevisiae that regulates the activity of the H(+)-ATPase. The cytoplasmic domain conformation results in a specific interfacial distribution of five basic side chains, thought to strongly interact with anionic phospholipids. We have used the PMP1 18-38 fragment to carry out a deuterium nuclear magnetic resonance ((2)H-NMR) study for investigating the interactions between the PMP1 cytoplasmic domain and phosphatidylserine...

  14. Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

    Energy Technology Data Exchange (ETDEWEB)

    Roche, Julien; Louis, John M.; Aniana, Annie [National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Chemical Physics (United States); Ghirlando, Rodolfo [National Institutes of Health, Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases (United States); Bax, Ad, E-mail: bax@nih.gov [National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Chemical Physics (United States)

    2015-04-15

    The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41’s ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.

  15. Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

    International Nuclear Information System (INIS)

    The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41’s ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion

  16. Melittin-Induced Bilayer Leakage Depends on Lipid Material Properties: Evidence for Toroidal Pores

    OpenAIRE

    Allende, Daniel; Simon, S. A.; McIntosh, Thomas J.

    2004-01-01

    The membrane-lytic peptide melittin has previously been shown to form pores in lipid bilayers that have been described in terms of two different structural models. In the “barrel stave” model the bilayer remains more or less flat, with the peptides penetrating across the bilayer hydrocarbon region and aggregating to form a pore, whereas in the “toroidal pore” melittin induces defects in the bilayer such that the bilayer bends sharply inward to form a pore lined by both peptides and lipid head...

  17. Rab3A is a new interacting partner of synaptotagmin I and may modulate synaptic membrane fusion through a competitive mechanism

    International Nuclear Information System (INIS)

    Highlights: • Rab3A has been found to be a novel interacting protein of synaptotagmin I. • Rab3A binds to synaptotagmin I in a Ca2+-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 Ca2+-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

  18. Lipid asymmetry in DLPC/DSPC supported lipid bilayers, a combined AFM and fluorescence microscopy study

    Energy Technology Data Exchange (ETDEWEB)

    Lin, W; Blanchette, C D; Ratto, T V; Longo, M L

    2005-06-20

    A fundamental attribute of cell membranes is transmembrane asymmetry, specifically the formation of ordered phase domains in one leaflet that are compositionally different from the opposing leaflet of the bilayer. Using model membrane systems, many previous studies have demonstrated the formation of ordered phase domains that display complete transmembrane symmetry but there have been few reports on the more biologically relevant asymmetric membrane structures. Here we report on a combined atomic force microscopy (AFM) and fluorescence microscopy study whereby we observe three different states of transmembrane symmetry in phase-separated supported bilayers formed by vesicle fusion. We find that if the leaflets differ in gel-phase area fraction, then the smaller domains in one leaflet are in registry with the larger domains in the other leaflet and the system is dynamic. In a presumed lipid flip-flop process similar to Ostwald Ripening, the smaller domains in one leaflet erode away while the large domains in the other leaflet grow until complete compositional asymmetry is reached and remains stable. We have quantified this evolution and determined that the lipid flip-flop event happens most frequently at the interface between symmetric and asymmetric DSPC domains. If both leaflets have nearly identical area fraction of gel-phase, gel-phase domains are in registry and are static in comparison to the first state. The stability of these three DSPC domain distributions, the degree of registry observed, and the domain immobility have direct biological significance with regards to maintenance of lipid asymmetry in living cell membranes, communication between inner leaflet and outer leaflet, membrane adhesion, and raft mobility.

  19. Regulation of sodium channel function by bilayer elasticity

    DEFF Research Database (Denmark)

    Lundbaek, Jens A; Birn, Pia; Hansen, Anker J;

    2004-01-01

    kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link a...... "conformational" change (the monomerdimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could...... be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-02-15

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

  1. Diffraction studies on natural and model lipid bilayers

    Science.gov (United States)

    Sebastiani, F.; Harvey, R.; Khanniche, S.; Artero, J.-B.; Haertlein, M.; Fragneto, G.

    2012-11-01

    In this study we have used neutron diffraction to examine the swelling behaviour and bilayer parameters of membranes reconstituted from polar lipids extracted from B. subtilis and model systems composed of synthetic phospholipids. Evidence for phase separation in the model system (lacking in Lysyl-PG, L-PG) is discussed in relation to its possible contribution to membrane domain formation through lipid-lipid interactions. Comparing these results with those obtained from the bilayers composed of lipids extracted from bacterial cells gives us some indication of the role of L-PG in the B. subtilis plasma membrane.

  2. Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization

    Directory of Open Access Journals (Sweden)

    Christian Kleusch

    2012-01-01

    Full Text Available In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.

  3. Millimeter-area, free standing, phospholipid bilayers.

    Science.gov (United States)

    Beltramo, Peter J; Van Hooghten, Rob; Vermant, Jan

    2016-05-11

    Minimal model biomembrane studies have the potential to unlock the fundamental mechanisms of cellular function that govern the processes upon which life relies. However, existing methods to fabricate free-standing model membranes currently have significant limitations. Bilayer sizes are often tens of micrometers, decoupling curvature or substrate effects, orthogonal control over tension, and solvent exchange combined with microscopy techniques is not possible, which restricts the studies that can be performed. Here, we describe a versatile platform to generate free standing, planar, phospholipid bilayers with millimeter scale areas. The technique relies on an adapted thin-film balance apparatus allowing for the dynamic control of the nucleation and growth of a planar black lipid membrane in the center of an orifice surrounded by microfluidic channels. Success is demonstrated using several different lipid types, including mixtures that show the same temperature dependent phase separation as existing protocols, moreover, membranes are highly stable. Two advantages unique to the proposed method are the dynamic control of the membrane tension and the possibility to make extremely large area membranes. We demonstrate this by showing how a block polymer, F68, used in drug delivery increases the membrane compliance. Together, the results demonstrate a new paradigm for studying the mechanics, structure, and function of model membranes. PMID:27050618

  4. Physisorbed Polymer-Tethered Lipid Bilayer with Lipopolymer Gradient

    Directory of Open Access Journals (Sweden)

    Christoph A. Naumann

    2012-11-01

    Full Text Available Physisorbed polymer-tethered lipid bilayers consisting of phospholipids and lipopolymers represent an attractive planar model membrane platform, in which bilayer fluidity and membrane elastic properties can be regulated through lipopolymer molar concentration. Herein we report a method for the fabrication of such a planar model membrane system with a lateral gradient of lipopolymer density. In addition, a procedure is described, which leads to a sharp boundary between regions of low and high lipopolymer molar concentrations. Resulting gradients and sharp boundaries are visualized on the basis of membrane buckling structures at elevated lipopolymer concentrations using epifluorescence microscopy and atomic force microscopy. Furthermore, results from spot photobleaching experiments are presented, which provide insight into the lipid lateral fluidity in these model membrane architectures. The presented experimental data highlight a planar, solid-supported membrane characterized by fascinating length scale-dependent dynamics and elastic properties with remarkable parallels to those observed in cellular membranes.

  5. A novel technique using hydrophilic polymers to promote axonal fusion

    Institute of Scientific and Technical Information of China (English)

    Ravinder Bamba; D Colton Riley; Nathaniel D Kelm; Mark D Does; Richard D Dortch; Wesley P hTayer

    2016-01-01

    The management of traumatic peripheral nerve injury remains a considerable concern for clinicians. With minimal innovations in surgical technique and a limited number of specialists trained to treat peripheral nerve injury, outcomes of surgical intervention have been unpredictable. The inability to manipulate the pathophysiology of nerve injury (i.e., Wallerian degeneration) has left scientists and clinicians depending on the slow and lengthy process of axonal regeneration (~1 mm/day). When axons are severed, the endings undergo calcium-mediated plasmalemmal sealing, which limits the ability of the axon to be primarily re-paired. Polythethylene glycol (PEG) in combination with a bioengineered process overcomes the inability to fuse axons. The mechanism for PEG axonal fusion is not clearly understood, but multiple studies have shown that a providing a calcium-free environment is essential to the process known as PEG fusion. The proposed mechanism is PEG-induced lipid bilayer fusion by removing the hydration barrier surrounding the axolemma and reducing the activation energy required for membrane fusion to occur. This review highlights PEG fusion, its past and current studies, and future directions in PEG fusion.

  6. Binding-Site Interactions between Epstein-Barr Virus Fusion Proteins gp42 and gH/gL Reveal a Peptide That Inhibits both Epithelial and B-Cell Membrane Fusion▿

    OpenAIRE

    Kirschner, Austin N.; Lowrey, Amanda S.; Longnecker, Richard; Theodore S Jardetzky

    2007-01-01

    Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N...

  7. A new automated technique for the reconstitution of hydrophobic proteins into planar bilayer membranes. Studies of human recombinant uncoupling protein 1

    Czech Academy of Sciences Publication Activity Database

    Beck, V.; Jabůrek, Martin; Breen, E. P.; Porter, R. K.; Ježek, Petr; Pohl, E. E.

    2006-01-01

    Roč. 1757, č. 5-6 (2006), s. 474-479. ISSN 0005-2728 R&D Projects: GA AV ČR(CZ) IAA5011106; GA MŠk(CZ) 1P05ME794 Grant ostatní: Deutsche Forschungsgemeinschaft(DE) Po-524/2-2; Deutsche Forschungsgemeinschaft(DE) 436 TSE 113/44/0-1 Institutional research plan: CEZ:AV0Z50110509 Keywords : artificial membranes * uncoupling protein-1 Subject RIV: BO - Biophysics Impact factor: 4.237, year: 2006

  8. Meet me on the other side: trans-bilayer modulation of a model voltage-gated ion channel activity by membrane electrostatics asymmetry.

    Directory of Open Access Journals (Sweden)

    Loredana Mereuta

    Full Text Available While it is accepted that biomembrane asymmetry is generated by proteins and phospholipids distribution, little is known about how electric changes manifested in a monolayer influence functional properties of proteins localized on the opposite leaflet. Herein we used single-molecule electrophysiology and investigated how asymmetric changes in the electrostatics of an artificial lipid membrane monolayer, generated oppositely from where alamethicin--a model voltage-gated ion channel--was added, altered peptide activity. We found that phlorizin, a membrane dipole potential lowering amphiphile, augmented alamethicin activity and transport features, whereas the opposite occurred with RH-421, which enhances the monolayer dipole potential. Further, the monolayer surface potential was decreased via adsorption of sodium dodecyl sulfate, and demonstrated that vectorial modification of it also affected the alamethicin activity in a predictive manner. A new paradigm is suggested according to which asymmetric changes in the monolayer dipole and surface potential extend their effects spatially by altering the intramembrane potential, whose gradient is sensed by distantly located peptides.

  9. Fabrication of Supported Lipid Bilayer on Graphene Oxide

    International Nuclear Information System (INIS)

    Planar lipid bilayers of dioleoylphosphatidylcholine was prepared on graphene oxide (GO), as a fundamental platform for biosensing in plasma membrane model using graphene. The GO flakes were prepared according to modified Hummer's method, and deposited on thermally oxidized SiO2/Si surfaces. We found that planar lipid bilayers were reproducibility formed on the GO/SiO2/Si surface in the presence of Ca2+ ions, while unruptured vesicles remained on the GO surface without Ca2+ ion. The results of atomic force microscope observation and fluorescence recovery after photobleaching experiment revealed that double lipid bilayers were spontaneously formed on the GO surfaces.

  10. Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

    OpenAIRE

    Bennett, W. F. Drew; Sapay, Nicolas; Tieleman, D. Peter

    2014-01-01

    Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DM...

  11. Induction of heterosubtypic cross-protection against influenza by a whole inactivated virus vaccine: the role of viral membrane fusion activity.

    Directory of Open Access Journals (Sweden)

    Natalija Budimir

    Full Text Available BACKGROUND: The inability of seasonal influenza vaccines to effectively protect against infection with antigenically drifted viruses or newly emerging pandemic viruses underlines the need for development of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. Therefore, potential cross-protective vaccines, e.g., whole inactivated virus (WIV vaccine, that can target conserved internal antigens such as the nucleoprotein (NP and/or matrix protein (M1 need to be explored. METHODOLOGY/PRINCIPAL FINDINGS: In the current study we show that a WIV vaccine, through induction of cross-protective cytotoxic T lymphocytes (CTLs, protects mice from heterosubtypic infection. This protection was abrogated after depletion of CD8+ cells in vaccinated mice, indicating that CTLs were the primary mediators of protection. Previously, we have shown that different procedures used for virus inactivation influence optimal activation of CTLs by WIV, most likely by affecting the membrane fusion properties of the virus. Specifically, inactivation with formalin (FA severely compromises fusion activity of the virus, while inactivation with β-propiolactone (BPL preserves fusion activity. Here, we demonstrate that vaccination of mice with BPL-inactivated H5N1 WIV vaccine induces solid protection from lethal heterosubtypic H1N1 challenge. By contrast, vaccination with FA-inactivated WIV, while preventing death after lethal challenge, failed to protect against development of disease and severe body weight loss. Vaccination with BPL-inactivated WIV, compared to FA-inactivated WIV, induced higher levels of specific CD8+ T cells in blood, spleen and lungs, and a higher production of granzyme B in the lungs upon H1N1 virus challenge. CONCLUSION/SIGNIFICANCE: The results underline the potential use of WIV as a cross-protective influenza vaccine candidate. However, careful choice of the virus inactivation procedure is important to retain membrane

  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.

    Directory of Open Access Journals (Sweden)

    Zhen Gong

    Full Text Available 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. An Rh1–GFP Fusion Protein Is in the Cytoplasmic Membrane of a White Mutant Strain of Chlamydomonas reinhardtii

    OpenAIRE

    Yoshihara, Corinne; Inoue, Kentaro; Schichnes, Denise; Ruzin, Steven; Inwood, William; Kustu, Sydney

    2008-01-01

    The major Rhesus (Rh) protein of the green alga Chlamydomonas reinhardtii, Rh1, is homologous to Rh proteins of humans. It is an integral membrane protein involved in transport of carbon dioxide. To localize a fusion of intact Rh1 to the green fluorescent protein (GFP), we used as host a white (lts1) mutant strain of C. reinhardtii, which is blocked at the first step of carotenoid biosynthesis. The lts1 mutant strain accumulated normal amounts of Rh1 heterotrophically in the dark and Rh1–GFP ...

  14. Compressibility of bilayer graphene

    OpenAIRE

    Borghi, Giovanni; Polini, Marco; Asgari, Reza; MacDonald, A. H.

    2010-01-01

    Bilayer graphene is a recently isolated and intriguing class of many-body systems with massive chiral quasiparticles. We present theoretical results for the electronic compressibility of bilayer graphene that are based on a four-band continuum band structure model combined with a random phase approximation treatment of electronic correlations. We find that the compressibility is strongly suppressed by electron-electron interactions at low carrier densities. Correlations do not lead to any qua...

  15. Protection of immunocompromised mice against lethal infection with Pseudomonas aeruginosa by active or passive immunization with recombinant P. aeruginosa outer membrane protein F and outer membrane protein I fusion proteins.

    OpenAIRE

    von Specht, B U; Knapp, B.; Muth, G; Bröker, M.; Hungerer, K D; Diehl, K D; Massarrat, K; Seemann, A; Domdey, H

    1995-01-01

    Recombinant outer membrane proteins (Oprs) of Pseudomonas aeruginosa were expressed in Escherichia coli as glutathione S-transferase (GST)-linked fusion proteins. GST-linked Oprs F and I (GST-OprF190-350 [GST linked to OprF spanning amino acids 190 to 350] and GST-OprI21-83, respectively) and recombinant hybrid Oprs (GST-OprF190-342-OprI21-83 and GST-OprI21-83-OprF190-350) were isolated and tested for their efficacy as vaccines in immunodeficient mice. GST-OprF-OprI protected the mice against...

  16. Structural Effects of Small Molecules on Phospholipid Bilayers Investigated by Molecular Simulations

    CERN Document Server

    Lee, B W; Sum, A K; Vattulainen, I; Patra, M; Karttunen, M; Lee, Bryan W; Faller, Roland; Sum, Amadeu K; Vattulainen, Ilpo; Patra, Michael; Karttunen, Mikko

    2004-01-01

    We summarize and compare recent Molecular Dynamics simulations on the interactions of dipalmitoylphosphatidylcholine (DPPC) bilayers in the liquid crystalline phase with a number of small molecules including trehalose, a disaccharide of glucose, alcohols, and dimethylsulfoxide (DMSO). The sugar molecules tend to stabilize the structure of the bilayer as they bridge adjacent lipid headgroups. They do not strongly change the structure of the bilayer. Alcohols and DMSO destabilize the bilayer as they increase its area per molecule in the bilayer plane and decrease the order parameter. Alcohols have a stronger detrimental effect than DMSO. The observables which we compare are the area per molecule in the plane of the bilayer, the membrane thickness, and the NMR order parameter of DPPC hydrocarbon tails. The area per molecule and the order parameter are very well correlated whereas the bilayer thickness is not necessarily correlated with them.

  17. Selective continuous monitoring and analysis of mixtures of acesulfame-K, cyclamate, and saccharin in artificial sweetener tablets, diet soft drinks, yogurts, and wines using filter-supported bilayer lipid membranes.

    Science.gov (United States)

    Nikolelis, D P; Pantoulias, S

    2001-12-15

    This work describes a technique for the rapid and sensitive electrochemical flow injection monitoring and analysis of mixtures of the artificial sweeteners acesulfame-K, cyclamate, and saccharin using stabilized systems of filter-supported bilayer lipid membranes (BLMs). Injections of artificial sweeteners were made into flowing streams of a carrier electrolyte solution, and a transient current signal with duration of seconds reproducibly appeared in less than sweeteners. The magnitude of this signal was linearly related to the concentration of artificial sweeteners, which could be determined at micromolar levels. Repetitive cycles of injection of artificial sweeteners have shown no signal degradation during each cycle (30 sequential injections). The time of appearance of the transient response was different for each artificial sweetener and increased in the order of cyclamic acid, acesulfame-K, and saccharin. The difference in time of response has allowed selective detection and analysis of these artificial sweeteners in mixtures. The effect of potent interferences, including a wide range of compounds usually found in foods, proteins, and lipids was investigated. The results showed no interferences from these constituents of real food samples. The major interference from proteins (most common in lipid-film-based biosensors) can be eliminated by modulation of the carrier solution that does not allow adsorption of these compounds in BLMs. The technique was applied in real food samples, that is, in artificial sweetener tablets, diet soft drinks, wines, and yogurts that contain mixtures of these artificial sweeteners with aspartame and other compounds. A comparison of results using the present method and that of an Official Method of Analysis showed good agreement between the two methods. PMID:11791564

  18. Lipid peroxidation and water penetration in lipid bilayers

    DEFF Research Database (Denmark)

    Conte, Elena; Megli, Francesco Maria; Khandelia, Himanshu;

    2012-01-01

    Lipid peroxidation plays a key role in the alteration of cell membrane's properties. Here we used as model systems multilamellar vesicles (MLVs) made of the first two products in the oxidative cascade of linoleoyl lecithin, namely 1-palmitoyl-2-(13-hydroperoxy-9,11-octadecanedienoyl)-lecithin (Hp......(zz) parameters revealed that OHPLPC, but mostly HpPLPC, induced a measurable increase in polarity and H-bonding propensity in the central region of the bilayer. Molecular dynamics simulation performed on 16-DSA in the PLPC-HpPLPC bilayer revealed that water molecules are statistically favored with respect to the...... hydroperoxide groups to interact with the nitroxide at the methyl-terminal, confirming that the H-bonds experimentally observed are due to increased water penetration in the bilayer. The EPR and MD data on model membranes demonstrate that cell membrane damage by oxidative stress cause alteration of water...

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

    Science.gov (United States)

    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. PMID:18852502

  20. Proteomics computational analyses suggest that the carboxyl terminal glycoproteins of Bunyaviruses are class II viral fusion protein (beta-penetrenes

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2004-11-01

    Full Text Available Abstract The Bunyaviridae family of enveloped RNA viruses includes five genuses, orthobunyaviruses, hantaviruses, phleboviruses, nairoviruses and tospoviruses. It has not been determined which Bunyavirus protein mediates virion:cell membrane fusion. Class II viral fusion proteins (beta-penetrenes, encoded by members of the Alphaviridae and Flaviviridae, are comprised of three antiparallel beta sheet domains with an internal fusion peptide located at the end of domain II. Proteomics computational analyses indicate that the carboxyl terminal glycoprotein (Gc encoded by Sandfly fever virus (SAN, a phlebovirus, has a significant amino acid sequence similarity with envelope protein 1 (E1, the class II fusion protein of Sindbis virus (SIN, an Alphavirus. Similar sequences and common structural/functional motifs, including domains with a high propensity to interface with bilayer membranes, are located collinearly in SAN Gc and SIN E1. Gc encoded by members of each Bunyavirus genus share several sequence and structural motifs. These results suggest that Gc of Bunyaviridae, and similar proteins of Tenuiviruses and a group of Caenorhabditis elegans retroviruses, are class II viral fusion proteins. Comparisons of divergent viral fusion proteins can reveal features essential for virion:cell fusion, and suggest drug and vaccine strategies.

  1. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

    International Nuclear Information System (INIS)

    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

  2. Pandemic H1N1 influenza A directly induces a robust and acute inflammatory gene signature in primary human bronchial epithelial cells downstream of membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Paquette, Stéphane G. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Institute of Medical Science, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Banner, David [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Chi, Le Thi Bao [Department of Microbiology, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Carlo Urbani Centre, Hue University of Medicine and Pharmacy, Thua Thien Hue (Viet Nam); Leon, Alberto J. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); Xu, Luoling; Ran, Longsi [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Huang, Stephen S.H. [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); Department of Immunology, Faculty of Medicine, University of Toronto, Toronto, Ontario (Canada); Farooqui, Amber [Division of Experimental Therapeutics, Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario (Canada); International Institute of Infection and Immunity, Shantou University Medical College, Shantou, Guangdong (China); 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. Determining the depth of insertion of dynamically invisible membrane peptides by gel-phase {sup 1}H spin diffusion heteronuclear correlation NMR

    Energy Technology Data Exchange (ETDEWEB)

    Wang, T.; Yao, H.; Hong, M., E-mail: mhong@iastate.edu [Iowa State University, Department of Chemistry (United States)

    2013-06-15

    Solid-state NMR determination of the depth of insertion of membrane peptides and proteins has so far utilized {sup 1}H spin diffusion and paramagnetic relaxation enhancement experiments, which are typically conducted in the liquid-crystalline phase of the lipid bilayer. For membrane proteins or peptide assemblies that undergo intermediate-timescale motion in the liquid-crystalline membrane, these approaches are no longer applicable because the protein signals are broadened beyond detection. Here we show that the rigid-solid HETCOR experiment, with an additional spin diffusion period, can be used to determine the depth of proteins in gel-phase lipid membranes, where the proteins are immobilized to give high-intensity solid-state NMR spectra. Demonstration on two membrane peptides with known insertion depths shows that well-inserted peptides give rise to high lipid cross peak intensities and low water cross peaks within a modest spin diffusion mixing time, while surface-bound peptides have higher water than lipid cross peaks. Furthermore, well-inserted membrane peptides have nearly identical {sup 1}H cross sections as the lipid chains, indicating equilibration of the peptide and lipid magnetization. Using this approach, we measured the membrane topology of the {alpha}-helical fusion peptide of the paramyxovirus, PIV5, in the anionic POPC/POPG membrane, in which the peptide undergoes intermediate-timescale motion at physiological temperature. The gel-phase HETCOR spectra indicate that the {alpha}-helical fusion peptide is well inserted into the POPC/POPG bilayer, spanning both leaflets. This insertion motif gives insight into the functional role of the {alpha}-helical PIV5 fusion peptide in virus-cell membrane fusion.

  4. Structural basis for membrane anchoring of HIV-1 envelope spike.

    Science.gov (United States)

    Dev, Jyoti; Park, Donghyun; Fu, Qingshan; Chen, Jia; Ha, Heather Jiwon; Ghantous, Fadi; Herrmann, Tobias; Chang, Weiting; Liu, Zhijun; Frey, Gary; Seaman, Michael S; Chen, Bing; Chou, James J

    2016-07-01

    HIV-1 envelope spike (Env) is a type I membrane protein that mediates viral entry. We used nuclear magnetic resonance to determine an atomic structure of the transmembrane (TM) domain of HIV-1 Env reconstituted in bicelles that mimic a lipid bilayer. The TM forms a well-ordered trimer that protects a conserved membrane-embedded arginine. An amino-terminal coiled-coil and a carboxyl-terminal hydrophilic core stabilize the trimer. Individual mutations of conserved residues did not disrupt the TM trimer and minimally affected membrane fusion and infectivity. Major changes in the hydrophilic core, however, altered the antibody sensitivity of Env. These results show how a TM domain anchors, stabilizes, and modulates a viral envelope spike and suggest that its influence on Env conformation is an important consideration for HIV-1 immunogen design. PMID:27338706

  5. Membrane Curvature in Flaviviruses

    OpenAIRE

    Zhang, Wei; Kaufmann, Bärbel; Chipman, Paul R.; Kuhn, Richard J; Rossmann, Michael G.

    2013-01-01

    Coordinated interplay between membrane proteins and the lipid bilayer is required for such processes as transporter function and the entrance of enveloped viruses into host cells. In this study, three-dimensional cryo-electron microscopy density maps of mature and immature flaviviruses were analyzed to assess the curvature of the membrane leaflets and its relation to membrane-bound viral glycoproteins. The overall morphology of the viral membrane is determined by icosahedral scaffolding compo...

  6. Cholesterol enhances surface water diffusion of phospholipid bilayers

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Chi-Yuan; Kausik, Ravinath; Han, Songi, E-mail: songi@chem.ucsb.edu [Department of Chemistry and Biochemistry and Materials Research Laboratory, University of California, Santa Barbara, California 93106 (United States); Olijve, Luuk L. C. [Laboratory of Macromolecular and Organic Chemistry and Institute for Complex Molecular Systems, Eindhoven University of Technology, P.O. Box 513, 5600 MB, Eindhoven (Netherlands)

    2014-12-14

    Elucidating the physical effect of cholesterol (Chol) on biological membranes is necessary towards rationalizing their structural and functional role in cell membranes. One of the debated questions is the role of hydration water in Chol-embedding lipid membranes, for which only little direct experimental data are available. Here, we study the hydration dynamics in a series of Chol-rich and depleted bilayer systems using an approach termed {sup 1}H Overhauser dynamic nuclear polarization (ODNP) NMR relaxometry that enables the sensitive and selective determination of water diffusion within 5–10 Å of a nitroxide-based spin label, positioned off the surface of the polar headgroups or within the nonpolar core of lipid membranes. The Chol-rich membrane systems were prepared from mixtures of Chol, dipalmitoyl phosphatidylcholine and/or dioctadecyl phosphatidylcholine lipid that are known to form liquid-ordered, raft-like, domains. Our data reveal that the translational diffusion of local water on the surface and within the hydrocarbon volume of the bilayer is significantly altered, but in opposite directions: accelerated on the membrane surface and dramatically slowed in the bilayer interior with increasing Chol content. Electron paramagnetic resonance (EPR) lineshape analysis shows looser packing of lipid headgroups and concurrently tighter packing in the bilayer core with increasing Chol content, with the effects peaking at lipid compositions reported to form lipid rafts. The complementary capability of ODNP and EPR to site-specifically probe the hydration dynamics and lipid ordering in lipid membrane systems extends the current understanding of how Chol may regulate biological processes. One possible role of Chol is the facilitation of interactions between biological constituents and the lipid membrane through the weakening or disruption of strong hydrogen-bond networks of the surface hydration layers that otherwise exert stronger repulsive forces, as reflected in

  7. Cholesterol enhances surface water diffusion of phospholipid bilayers

    Science.gov (United States)

    Cheng, Chi-Yuan; Olijve, Luuk L. C.; Kausik, Ravinath; Han, Songi

    2014-12-01

    Elucidating the physical effect of cholesterol (Chol) on biological membranes is necessary towards rationalizing their structural and functional role in cell membranes. One of the debated questions is the role of hydration water in Chol-embedding lipid membranes, for which only little direct experimental data are available. Here, we study the hydration dynamics in a series of Chol-rich and depleted bilayer systems using an approach termed 1H Overhauser dynamic nuclear polarization (ODNP) NMR relaxometry that enables the sensitive and selective determination of water diffusion within 5-10 Å of a nitroxide-based spin label, positioned off the surface of the polar headgroups or within the nonpolar core of lipid membranes. The Chol-rich membrane systems were prepared from mixtures of Chol, dipalmitoyl phosphatidylcholine and/or dioctadecyl phosphatidylcholine lipid that are known to form liquid-ordered, raft-like, domains. Our data reveal that the translational diffusion of local water on the surface and within the hydrocarbon volume of the bilayer is significantly altered, but in opposite directions: accelerated on the membrane surface and dramatically slowed in the bilayer interior with increasing Chol content. Electron paramagnetic resonance (EPR) lineshape analysis shows looser packing of lipid headgroups and concurrently tighter packing in the bilayer core with increasing Chol content, with the effects peaking at lipid compositions reported to form lipid rafts. The complementary capability of ODNP and EPR to site-specifically probe the hydration dynamics and lipid ordering in lipid membrane systems extends the current understanding of how Chol may regulate biological processes. One possible role of Chol is the facilitation of interactions between biological constituents and the lipid membrane through the weakening or disruption of strong hydrogen-bond networks of the surface hydration layers that otherwise exert stronger repulsive forces, as reflected in faster

  8. Fusion behaviour of aquaporin Z incorporated proteoliposomes investigated by quartz crystal microbalance with dissipation (QCM-D).

    Science.gov (United States)

    Li, Xuesong; Wang, Rong; Wicaksana, Filicia; Zhao, Yang; Tang, Chuyang; Torres, Jaume; Fane, Anthony Gordon

    2013-11-01

    Aquaporin-based biomimetic membranes have potential as promising membranes for water purification and desalination due to the exceptionally high water permeability and selectivity of aquaporins. However, the design and preparation of such membranes for practical applications are very challenging as the relevant fundamental research is rather limited to provide guidance. Here we investigated the basic characteristics and fusion behaviour of proteoliposomes incorporated with aquaporin Z (AqpZ) on to solid surfaces. This study is expected to offer a better understanding of the properties of proteoliposomes and the potential of the vesicle fusion technique. Our results show that after incorporation of AqpZ, the size and surface charge density of the proteoliposomes change significantly compared with those of liposomes. Although the liposome could easily form a supported lipid bilayer on silica via vesicle rupture, it is much more difficult for proteoliposomes to fuse completely into a bilayer on the same substrate. In addition, the fusion of proteoliposomes is further hindered as the density of incorporated AqpZ is increased, suggesting that proteoliposome with more proteins become more robust. However, both the liposome and proteoliposome have difficulty forming supported lipid bilayers on the surface of a polyelectrolyte layer even though it carries an opposite charge, indicating that the polymer may play an important role in stabilising vesicles. It was also observed that a high concentration of AqpZ could be incorporated into the 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) liposome even though its permeability decreased. These findings may provide some useful guidance for preparing such biomimetic membranes. PMID:23850749

  9. Mesoscopic models of biological membranes

    DEFF Research Database (Denmark)

    Venturoli, M.; Sperotto, Maria Maddalena; Kranenburg, M.;

    2006-01-01

    Phospholipids are the main components of biological membranes and dissolved in water these molecules self-assemble into closed structures, of which bilayers are the most relevant from a biological point of view. Lipid bilayers are often used, both in experimental and by theoretical investigations...

  10. Lipid Bilayer – mediated Regulation of Ion Channel Function by Amphiphilic Drugs

    DEFF Research Database (Denmark)

    Lundbæk, Jens August

    2008-01-01

    that are transforming it into a subject of quantitative science. It is described how the hydrophobic interactions between a membrane protein and the host lipid bilayer provide the basis for a mechanism, whereby protein function is regulated by the bilayer physical properties. The use of gramicidin...

  11. Dynamic Structure Formation of Peripheral Membrane Proteins

    OpenAIRE

    Morozova, Diana; Guigas, Gernot; Weiss, Matthias

    2011-01-01

    Author Summary Eukaryotic cells are subdivided into a variety of compartments by membranes, i.e. by lipid bilayers into which a multitude of proteins are embedded. About 30% of all protein species in a cell are associated with membranes to perform vital functions, e.g. in signaling and transport pathways. A plethora of membrane-associated proteins, so-called peripheral membrane proteins, penetrate only one monolayer whereas transmembrane proteins span the entire thickness of a lipid bilayer. ...

  12. Interfacing Zwitterionic Liposomes with Inorganic Nanomaterials: Surface Forces, Membrane Integrity, and Applications.

    Science.gov (United States)

    Liu, Juewen

    2016-05-10

    Zwitterionic phosphocholine (PC) lipids are the main constituent of the mammalian cell membrane. PC bilayers are known for their antifouling properties, yet they are adsorbed by all tested inorganic nanoparticles. This feature article is focused on the developments in my laboratory in the past few years on this topic. The main experimental techniques include fluorescence-based liposome leakage assays, adsorption and desorption, and cryo-TEM. Different materials interact with PC liposomes differently. PC liposomes adsorb on SiO2, followed by membrane fusion with the surface forming supported lipid bilayers. TiO2 and other metal oxides adsorb only intact PC liposomes via lipid phosphate bonding; the steric effect from the choline group hinders subsequent liposome fusion onto the particles. Citrate-capped AuNPs are adsorbed very strongly via van der Waals forces, inducing local gelation. The result is transient liposome leakage upon AuNP adsorption or desorption and AuNP aggregation on the liposome surface. All carbon-based nanomaterials (graphene oxides, carbon nanotubes, and nanodiamond) are adsorbed mainly via hydrogen bonding. The oxidation level of graphene oxide strongly influences the outcome of the final hybrid material. In the context of inorganic nanoparticle adsorption, insights are given regarding the lack of protein adsorption by PC bilayers. These inorganic/lipid hybrid materials can be used for controlled release, drug delivery, and fundamental studies. A few examples of application are covered toward the end, and future perspectives are given. PMID:27093351

  13. Fluorescence and atomic force microscopy to visualize the interaction of HDL particles with lipid membranes

    International Nuclear Information System (INIS)

    Full text: High density lipoprotein (HDL) plays a key role in cholesterol homeostasis: cholesterol-loaded HDL particles are transported from non-hepatic peripheral tissue to the liver, where they unload their cargo via receptor-mediated selective uptake. It is astonishing that – although blood levels of HDL are broadly used in diagnosis for the prognosis of developing cardiovascular disease – the cholesterol uptake mechanisms are still poorly understood. Particularly, it remains unclear how the amphipathic cholesterol crosses the aqueous phase between the HDL particle and the cell membrane. We applied state-of-the-art high-resolution and ultra-sensitive force and fluorescence microscopy techniques to image directly the interaction of HDL particles with the target membrane. Using highspeed atomic-force microscopy (AFM) we made a surprising discovery: when added to membranes, we observed HDL particles to integrate into the interleaflet core of the bilayer, generating 'nanoblisters' with a size below 10 nm. Amphipathic cargo was able to leave such blisters, whereas hydrophobic cargo such as cholesteryl-ester remained associated with the particles. Using a combined fluorescence and force microscopy system we could directly visualize the transfer of single cargo molecules into supported lipid bilayers. Particularly, we compared the transfer of the fluorescently labelled amphiphilic DiI and Bodipy-labelled cholesterol with the hydrophobic Bodipy-labelled cholesteryl- ester. Our experiments revealed that i) cargo transfer requires contact; ii) only amphiphilic cargo is transferred. Interestingly, membrane elasticity was found to be crucial for the fusion: only highly elastic (and thus cholesterol-poor) membranes facilitate particle fusion, whereas inelastic cholesterol-rich membranes prevented the fusion. Live cell experiments show that the plasma membrane itself regulates a cell’s cholesterol demand: high cholesterol levels act repulsive, low cholesterol levels

  14. Membrane protein structure determination: back to the membrane.

    Science.gov (United States)

    Yao, Yong; Ding, Yi; Tian, Ye; Opella, Stanley J; Marassi, Francesca M

    2013-01-01

    NMR spectroscopy enables the structures of membrane proteins to be determined in the native-like environment of the phospholipid bilayer membrane. This chapter outlines the methods for membrane protein structural studies using solid-state NMR spectroscopy with samples of membrane proteins incorporated in proteoliposomes or planar lipid bilayers. The methods for protein expression and purification, sample preparation, and NMR experiments are described and illustrated with examples from OmpX and Ail, two bacterial outer membrane proteins that function in bacterial virulence. PMID:23975776

  15. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    International Nuclear Information System (INIS)

    E2, along with Erns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, 818CPIGWTGVIEC828, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP 818CPIGWTGVIEC828 indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion

  16. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Fernández-Sainz, I.J. [Plum Island Animal Disease Center, ARS, USDA (United States); Largo, E. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Gladue, D.P.; Fletcher, P. [Plum Island Animal Disease Center, ARS, USDA (United States); O’Donnell, V. [Plum Island Animal Disease Center, ARS, USDA (United States); Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Holinka, L.G. [Plum Island Animal Disease Center, ARS, USDA (United States); Carey, L.B. [Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), E-08003 Barcelona (Spain); Lu, X. [Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Nieva, J.L. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Borca, M.V., E-mail: manuel.borca@ars.usda.gov [Plum Island Animal Disease Center, ARS, USDA (United States)

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  17. Crystal Structure of a Soluble Fragment of the Membrane Fusion Protein HlyD in a Type I Secretion System of Gram-Negative Bacteria.

    Science.gov (United States)

    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. PMID:26833388

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Stability of charged membranes

    OpenAIRE

    Bensimon, D; David, F.; Leibler, S.; Pumir, A.

    1990-01-01

    The electrostatic contribution to the bending elastic modulus of charged phospholipid bilayers in an ionic solution is computed. It is found to be the same for conducting and non-conducting membranes and is always stabilizing. This stability for free membranes is shown to be a simple consequence of the vanishing of the physical surface tension.

  20. Influence of membrane composition on its flexibility

    International Nuclear Information System (INIS)

    Full text: Lamellar phases and vesicles composed of lipids have been used as model systems to investigate biological process related to cell membrane as well as promising carriers for drugs and gene therapy. The composition of the membrane determines its three dimensional shape and its properties such as rigidity and compressibility which play an important role on membrane fusion, protein adhesion, interactions between proteins, etc. We present systematic study of a lamellar system composed mainly of lecithin which is a biocompatible phospholipid and simusol, which is a mixture of fatty acids that acts as a cosurfactant introducing flexibility to the membrane. Using X ray scattering we determine the lamellar periodicity as a function of the hydration for different formulations of the membrane; ranging from 100 % to 50 % mass fraction of lecithin. The X-ray spectra are fitted using a 4 Gaussian model [1]that allows us to determine the lamellar periodicity and the Caille parameter [2]. The ideal swelling law relating the membrane volume fraction (φm) to the lamellar periodicity (D) is given by φm =δm/D, where δm is the thickness membrane, however, when steric interactions are dominant with respect to electrostatic and van der Waals interactions, deviations from this behavior are expected [3]. We present experimental data illustrating the swelling behavior for the membrane compositions and the respective behavior of the hydration limit, membrane Luzzati [4], of the Caille parameter and qualitative interpretation of the interaction forces the systems studying the parameter membrane square amplitude fluctuation[5]. [1] Private communication with Prof. Dr. Cristiano Luis Pinto de Oliveira. [2] Caille A. et all, Acad. Sci. Paris B274 (1972) 891. [3] E. Kurtisovski et all, PRL 98, 258103 (2007). [4] Nagle et all, Curr Opin Struct Biol. 2000 Aug;10(4):474-80. [5] H. I. Petrache. Structure and interactions of fluid phospholipids bilayers measured by high resolution X

  1. Neutron spin echo study on elastic properties of phospholipid membranes interacting with pore-forming peptides

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hwan

    2010-02-15

    Cell membranes, which consist of phospholipid bilayers, play important roles in cells as barriers for maintaining concentrations and matrices to host membrane proteins. During cellular processes such as endo- and exo-cytosis, cell fission and fusion, the cell membranes undergo various morphological changes which are mainly governed by the interplay between protein and lipid membranes. A number of mechanisms have been proposed for protein-induced membrane deformations, including insertion of amphipathic helices, direct or indirect scafolding, and oligomerization of membrane proteins which change membrane curvature. However, it is not well exploited how the elastic properties of membranes, which play a key role in membrane deformation, are affected by the protein-membrane interactions. Therefore, to understand cell functions related protein-membrane interactions, in terms of the elastic properties of the membrane, is an important step toward elucidating the mechanisms of protein functions in membranes. A well-known example of protein-membrane interaction is the activity of antimicrobial peptides. Such peptides associate with a lipid bilayer in two distinct ways. At low peptide to lipid molar ratio P/L, the peptides adsorb horizontally to the surface of membrane and above a threshold concentration P/L*, the peptides begin to insert into the membrane, forming trans-membrane pores. Here, we report, for the first time, the thermal fuctuation and elasticity of dioleoyl phosphocholine large unilamellar vesicle membranes interacting with pore-forming peptides, melittin, which were measured by in-situ neutron spin echo spectroscopy (NSE). The relaxation behavior of the intermediate dynamic structure factors of the membrane at different P/L can be divided into three regions, resulting from characteristic changes of the effective bending modulus Κ(Κ = +d{sup 2}k, where Κ is the bending modulus, d is the height of the neutral surface from bilayer midplane and k is the

  2. Positively charged supported lipid bilayer formation on gold surfaces for neuronal cell culture.

    Science.gov (United States)

    Choi, Sung-Eun; Greben, Kyrylo; Wördenweber, Roger; Offenhäusser, Andreas

    2016-06-01

    Supported lipid bilayers are widely used as cell membrane models and sensor platforms, but the usage on gold surface needs additional surface modification or optimized experimental conditions. In this work, the authors show lipid bilayer formation on plasma activated gold surfaces in physiological conditions without any other modification if at least 30% positively charged lipids are present. Details of bilayer formation from small unilamellar vesicles were monitored using quartz crystal microbalance with dissipation in both basic and acidic environment. The authors also confirmed that this positively charged bilayer system can sustain primary cortical neuron growth and lipid transfer. This method will provide simple means to construct biomimetic interface on gold electrodes. PMID:27052005

  3. Interaction of a synthetic peptide corresponding to the N-terminus of canine distemper virus fusion protein with phospholipid vesicles: a biophysical study.

    Science.gov (United States)

    Aranda, Francisco J; Teruel, José A; Ortiz, Antonio

    2003-12-01

    region of the fluid bilayer. FTIR studies of the Cz=O stretching band indicated that incorporation of FP31 into phosphatidylcholine membranes produced a strong dehydration of the polar part of the bilayer. In mixtures with L-alpha-dielaidoylphosphatidylethanolamine (DEPE), increasing FP31 concentrations broadened and shifted to lower temperatures the lamellar to hexagonal-HII phase transition, indicating that this peptide destabilized the bilayer and promoted formation of the hexagonal-HII phase. The results are discussed in terms of lipid-peptide hydrophobic mismatch and its influence on the role of the N-terminal polypeptide of CDV F1 protein in viral membrane fusion. PMID:14643933

  4. Studying the Nucleated Mammalian Cell Membrane by Single Molecule Approaches

    Science.gov (United States)

    Wang, Feng; Wu, Jiazhen; Gao, Jing; Liu, Shuheng; Jiang, Junguang; Jiang, Shibo; Wang, Hongda

    2014-01-01

    The cell membrane plays a key role in compartmentalization, nutrient transportation and signal transduction, while the pattern of protein distribution at both cytoplasmic and ectoplasmic sides of the cell membrane remains elusive. Using a combination of single-molecule techniques, including atomic force microscopy (AFM), single molecule force spectroscopy (SMFS) and stochastic optical reconstruction microscopy (STORM), to study the structure of nucleated cell membranes, we found that (1) proteins at the ectoplasmic side of the cell membrane form a dense protein layer (4 nm) on top of a lipid bilayer; (2) proteins aggregate to form islands evenly dispersed at the cytoplasmic side of the cell membrane with a height of about 10–12 nm; (3) cholesterol-enriched domains exist within the cell membrane; (4) carbohydrates stay in microdomains at the ectoplasmic side; and (5) exposed amino groups are asymmetrically distributed on both sides. Based on these observations, we proposed a Protein Layer-Lipid-Protein Island (PLLPI) model, to provide a better understanding of cell membrane structure, membrane trafficking and viral fusion mechanisms. PMID:24806512

  5. Neutron diffraction studies of amphipathic helices in phospholipid bilayers

    International Nuclear Information System (INIS)

    The structural feature which is thought to facilitate the interaction of many peptides with phospholipid bilayers is the ability to fold into an amphipathic helix. In most cases the exact location and orientation of this helix with respect to the membrane is not known, and may vary with factors such as pH and phospholipid content of the bilayer. The growing interest in this area is stimulated by indications that similar interactions can contribute to the binding of certain hormones to their cell-surface receptors. We have been using the techniques of neutron diffraction from stacked phospholipid bilayers in an attempt to investigate this phenomenon with a number of membrane-active peptides. Here we report some of our findings with three of these: the bee venom melittin; the hormone calcitonin; and a synthetic peptide representing the ion channel fragment of influenza A M2 protein

  6. Neutron diffraction studies of amphipathic helices in phospholipid bilayers

    Energy Technology Data Exchange (ETDEWEB)

    Bradshaw, J.P.; Gilchrist, P.J. [Univ. of Edinburgh (United Kingdom); Duff, K.C. [Univ. of Edinburgh Medical School (United Kingdom); Saxena, A.M. [Brookhaven National Laboratory, Upton, NY (United States)

    1994-12-31

    The structural feature which is thought to facilitate the interaction of many peptides with phospholipid bilayers is the ability to fold into an amphipathic helix. In most cases the exact location and orientation of this helix with respect to the membrane is not known, and may vary with factors such as pH and phospholipid content of the bilayer. The growing interest in this area is stimulated by indications that similar interactions can contribute to the binding of certain hormones to their cell-surface receptors. We have been using the techniques of neutron diffraction from stacked phospholipid bilayers in an attempt to investigate this phenomenon with a number of membrane-active peptides. Here we report some of our findings with three of these: the bee venom melittin; the hormone calcitonin; and a synthetic peptide representing the ion channel fragment of influenza A M2 protein.

  7. Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

    Energy Technology Data Exchange (ETDEWEB)

    Drücker, Patrick [Institute of Biochemistry, University of Münster, Wilhelm-Klemm-Str. 2, D-48149 Münster (Germany); Gerke, Volker [Institute of Medical Biochemistry, ZMBE, University of Münster, Von-Esmarch-Str. 56, D-48149 Münster (Germany); Galla, Hans-Joachim, E-mail: gallah@uni-muenster.de [Institute of Biochemistry, University of Münster, Wilhelm-Klemm-Str. 2, D-48149 Münster (Germany)

    2014-10-10

    Highlights: • We show long-term mechanical stabilization of solid supported bilayers. • Bilayer integrity is essential for the investigation of protein–lipid interactions. • Protein adsorption to a bilayer containing defects causes membrane destruction. - Abstract: The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO{sub 2} and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P{sub 2} (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P{sub 2} and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications.

  8. Importance of phospholipid bilayer integrity in the analysis of protein–lipid interactions

    International Nuclear Information System (INIS)

    Highlights: • We show long-term mechanical stabilization of solid supported bilayers. • Bilayer integrity is essential for the investigation of protein–lipid interactions. • Protein adsorption to a bilayer containing defects causes membrane destruction. - Abstract: The integrity of supported phospholipid bilayer membranes is of crucial importance for the investigation of lipid–protein interactions. Therefore we recorded the formation of supported membranes on SiO2 and mica by quartz crystal microbalance and controlled the integrity by atomic force microscopy. This study aims to analyze how membrane defects affect protein–lipid interactions. The experiments focused on a lipid mixture of POPC/DOPC/Chol/POPS/PI(4,5)P2 (37:20:20:20:3) and the binding of the peripheral membrane associated protein annexin A2. We found that formation of a continuous undisturbed bilayer is an indispensable precondition for a reliable determination and quantification of lipid–protein-interactions. If membrane defects were present, protein adsorption causes membrane disruption and lipid detachment on a support thus leading to false determination of binding constants. Our results obtained for PI(4,5)P2 and cholesterol containing supported membranes yield new knowledge to construct functional surfaces that may cover nanoporous substrates, form free standing membranes or may be used for lab-on-a-chip applications

  9. Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

    OpenAIRE

    Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Devoisselle, Jean-Marie; Ladant, Daniel; Chopineau, Joel

    2013-01-01

    Many bacterial toxins can cross biological membranes to reach the cytosol of mammalian cells, although how they pass through a lipid bilayer remains largely unknown. Bordetella pertussis adenylate cyclase (CyaA) toxin delivers its catalytic domain directly across the cell membrane. To characterize this unique translocation process, we designed an in vitro assay based on a tethered lipid bilayer assembled over a biosensor surface derivatized with calmodulin, a natural activator of the toxin. C...

  10. High Resistivity Lipid Bilayers Assembled on Polyelectrolyte Multilayer Cushions: An Impedance Study.

    Science.gov (United States)

    Diamanti, Eleftheria; Gregurec, Danijela; Rodríguez-Presa, María José; Gervasi, Claudio A; Azzaroni, Omar; Moya, Sergio E

    2016-06-28

    Supported membranes on top of polymer cushions are interesting models of biomembranes as cell membranes are supported on a polymer network of proteins and sugars. In this work lipid vesicles formed by a mixture of 30% 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 70% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) are assembled on top of a polyelectrolyte multilayer (PEM) cushion of poly(allylamine hydrochloride) (PAH) and poly(styrene sodium sulfonate) (PSS). The assembly results in the formation of a bilayer on top of the PEM as proven by means of the quartz crystal microbalance with dissipation technique (QCM-D) and by cryo-transmission electron microscopy (cryo-TEM). The electrical properties of the bilayer are studied by electrochemical impedance spectroscopy (EIS). The bilayer supported on the PEMs shows a high resistance, on the order of 10(7) Ω cm(2), which is indicative of a continuous, dense bilayer. Such resistance is comparable with the resistance of black lipid membranes. This is the first time that such values are obtained for lipid bilayers supported on PEMs. The assembly of polyelectrolytes on top of a lipid bilayer decreases the resistance of the bilayer up to 2 orders of magnitude. The assembly of the polyelectrolytes on the lipids induces defects or pores in the bilayer which in turn prompts a decrease in the measured resistance. PMID:27267089

  11. Manipulating lipid membrane architecture by liquid crystal-analog curvature elasticity (Presentation Recording)

    Science.gov (United States)

    Lee, Sin-Doo

    2015-10-01

    Soft matters such as liquid crystals and biological molecules exhibit a variety of interesting physical phenomena as well as new applications. Recently, in mimicking biological systems that have the ability to sense, regulate, grow, react, and regenerate in a highly responsive and self-adaptive manner, the significance of the liquid crystal order in living organisms, for example, a biological membrane possessing the lamellar order, is widely recognized from the viewpoints of physics and chemistry of interfaces and membrane biophysics. Lipid bilayers, resembling cell membranes, provide primary functions for the transport of biological components of ions and molecules in various cellular activities, including vesicle budding and membrane fusion, through lateral organization of the membrane components such as proteins. In this lecture, I will describe how the liquid crystal-analog curvature elasticity of a lipid bilayer plays a critical role in developing a new platform for understanding diverse biological functions at a cellular level. The key concept is to manipulate the local curvature at an interface between a solid substrate and a model membrane. Two representative examples will be demonstrated: one of them is the topographic control of lipid rafts in a combinatorial array where the ligand-receptor binding event occurs and the other concerns the reconstitution of a ring-type lipid raft in bud-mimicking architecture within the framework of the curvature elasticity.

  12. 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

    Science.gov (United States)

    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

  13. A Molecular Dynamics Study of the Structural and Dynamical Properties of Putative Arsenic Substituted Lipid Bilayers

    Directory of Open Access Journals (Sweden)

    Ratna Juwita

    2013-04-01

    Full Text Available Cell membranes are composed mainly of phospholipids which are in turn, composed of five major chemical elements: carbon, hydrogen, nitrogen, oxygen, and phosphorus. Recent studies have suggested the possibility of sustaining life if the phosphorus is substituted by arsenic. Although this issue is still controversial, it is of interest to investigate the properties of arsenated-lipid bilayers to evaluate this possibility. In this study, we simulated arsenated-lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-arsenocholine (POAC, lipid bilayers using all-atom molecular dynamics to understand basic structural and dynamical properties, in particular, the differences from analogous 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, (POPC lipid bilayers. Our simulations showed that POAC lipid bilayers have distinct structural and dynamical properties from those of native POPC lipid bilayers. Relative to POPC lipid bilayers, POAC lipid bilayers have a more compact structure with smaller lateral areas and greater order. The compact structure of POAC lipid bilayers is due to the fact that more inter-lipid salt bridges are formed with arsenate-choline compared to the phosphate-choline of POPC lipid bilayers. These inter-lipid salt bridges bind POAC lipids together and also slow down the head group rotation and lateral diffusion of POAC lipids. Thus, it would be anticipated that POAC and POPC lipid bilayers would have different biological implications.

  14. Large scale biomimetic membrane arrays

    DEFF Research Database (Denmark)

    Hansen, Jesper Søndergaard; Perry, Mark; Vogel, Jörg;

    2009-01-01

    peptides and proteins. Next, we tested the scalability of the biomimetic membrane design by establishing lipid bilayers in rectangular 24 x 24 and hexagonal 24 x 27 aperture arrays, respectively. The results presented show that the design is suitable for further developments of sensitive biosensor assays......To establish planar biomimetic membranes across large scale partition aperture arrays, we created a disposable single-use horizontal chamber design that supports combined optical-electrical measurements. Functional lipid bilayers could easily and efficiently be established across CO2 laser micro......, and furthermore demonstrate that the design can conveniently be scaled up to support planar lipid bilayers in large square-centimeter partition arrays....

  15. Formation of artificial lipid bilayers using droplet dielectrophoresis

    OpenAIRE

    Aghdaei, Sara; Sandison, Mairi E.; Zagnoni, Michele; Green, Nicolas G; Morgan, Hywel

    2008-01-01

    We describe the formation of artificial bilayer lipid membranes (BLMs) by the controlled, electrical manipulation of aqueous droplets immersed in a lipid-alkane solution. Droplet movement was generated using dielectrophoresis on planar microelectrodes covered in a thin insulator. Droplets, surrounded by lipid monolayers, were brought into contact and spontaneously formed a BLM. The method produced BLMs suitable for single-channel recording of membrane protein activity and the technique can be...

  16. Atomic force microscopic study on lipid bilayer nanoscale phase separation

    OpenAIRE

    Liu, Rui

    2014-01-01

    Phase separation of copolymers or lipid membranes in nanoscale has attracted increasing interests for their applications in the synthesis of inorganic nanomaterial. The nanoscale phase separation of liquid bilayer as a supported membrane is systematically investigated by atomic force microscope (AFM). Moreover, the position of the fluorescence indicator, which is commonly used in the phase separation study in optical microscope, is also probed in this study to complete the knowledge of tradit...

  17. The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes.

    Science.gov (United States)

    Chlanda, Petr; Mekhedov, Elena; Waters, Hang; Schwartz, Cindi L; Fischer, Elizabeth R; Ryham, Rolf J; Cohen, Fredric S; Blank, Paul S; Zimmerberg, Joshua

    2016-01-01

    Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion(1,2). For viral fusion, the hemifusion structures are not determined(3). Here, influenza virus-like particles(4) carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S(5) and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a 'lipidic junction'. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a rupture-insertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis. PMID:27572837

  18. The effects of cryosolvents on DOPC-β-sitosterol bilayers determined from molecular dynamics simulations.

    Science.gov (United States)

    Hughes, Zak E; Malajczuk, Chris J; Mancera, Ricardo L

    2013-03-28

    Polyhydroxylated alcohols and DMSO are common cryosolvents that can damage cell membranes at sufficiently high concentrations. The interaction of representative plant cell membranes composed of mixed 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)-β-sitosterol bilayers, at a range of compositions, with a variety of cryosolvent molecules (DMSO, propylene glycol, ethylene glycol, glycerol, and methanol) has been investigated using molecular dynamics simulations. All the cryosolvents cause the bilayers to thin and become disordered; however, DMSO and propylene glycol have a greater disordering effect on the bilayer. Propylene glycol is shown to have the ability to cause the formation of pores in pure DOPC bilayers in a manner similar to that previously shown for DMSO. As the concentration of β-sitosterol within the bilayer increases, the membranes become more resistant to the deleterious effects of the cryosolvents. All three polyhydroxylated species are observed to form hydrogen bonds to multiple phospholipid molecules, effectively acting as cross-linkers, with glycerol being the most effective cross-linker. Increases in the concentration of β-sitosterol reduce overall hydrogen bonding of the bilayer with the cryosolvents as well as cross-linking, with glycerol and ethylene glycol being the most affected. The ability of all of these cryosolvents to affect the integrity of cell membranes appears to be the result of the balance of their ability to disorder lipid bilayers, to diffuse across them, and to interact with the lipid head groups. PMID:23445456

  19. Microporous device for local electric recordings on model lipid bilayers

    International Nuclear Information System (INIS)

    A powerful approach for characterizing lipid membranes and embedded proteins is the reconstitution of model lipid bilayers. The extreme fragility of 5 nm thick bilayers is a challenge for device design and requires a trade off of stability against accessibility. We here present a microporous lab-on-chip device that allows us to form stable, solvent-free lipid bilayers from giant unilamellar vesicles (GUVs) in a geometry that provides a unique set of access possibilities. The device is constructed around a micro-fabricated silicon chip with clusters of 1 µm-diameter pores and provides optical access to the lipid bilayers for high-NA epifluorescence imaging. At the same time, solvent exchange is possible on both sides of the lipid bilayer. Complete coverage can be achieved with GUVs, so that voltages can be applied across the lipid bilayer and single-channel currents can be measured using external or integrated silver/silver chloride electrodes. We describe the micro-fabrication by standard cleanroom techniques and the characterization of the device by atomic force microscopy, scanning electron microscopy and impedance spectroscopy. In proof-of-concept experiments we demonstrate that the device is capable of low-noise, single-ion-channel recordings. (paper)

  20. 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.

    Science.gov (United States)

    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