Nanoscopic Electrofocusing for Bio-Nanoelectronic Devices

Science.gov (United States)

Lakshmanan, Shanmugamurthy

2015-01-01

The ability to arrange precisely designed patterns of nanoparticles into a desired spatial configuration is the key to creating novel nanoscale devices that take advantage of the unique properties of nanomaterials. While two-dimensional arrays of nanoparticles have been demonstrated successfully by various techniques, a controlled way of building ordered arrays of three-dimensional (3D) nanoparticle structures remains challenging. This book describes a new technique called the 'nanoscopic lens' which is able to produce a variety of 3D nano-structures in a controlled manner. This ebook describes the nanoscopic lens technique and how it can serve as the foundation for device development that is not limited to a variety of optical, magnetic and electronic devices, but can also create a wide range of bio-nanoelectronic devices.

Superdiffusive motion of membrane-targeting C2 domains

Science.gov (United States)

Campagnola, Grace; Nepal, Kanti; Schroder, Bryce W.; Peersen, Olve B.; Krapf, Diego

2015-12-01

Membrane-targeting domains play crucial roles in the recruitment of signalling molecules to the plasma membrane. For most peripheral proteins, the protein-to-membrane interaction is transient. After proteins dissociate from the membrane they have been observed to rebind following brief excursions in the bulk solution. Such membrane hops can have broad implications for the efficiency of reactions on membranes. 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 behaviour. However, traditional time-averaged MSD analysis of individual trajectories remains linear and does not reveal superdiffusion. Our observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution. These hopping events allow proteins to explore large areas in a short time. The experimental results are shown to be consistent with analytical models of bulk-mediated diffusion and numerical simulations.

Yeast lipids can phase separate into micrometer-scale membrane domains

DEFF Research Database (Denmark)

Klose, Christian; Ejsing, Christer S; Garcia-Saez, Ana J

2010-01-01

The lipid raft concept proposes that biological membranes have the potential to form functional domains based on a selective interaction between sphingolipids and sterols. These domains seem to be involved in signal transduction and vesicular sorting of proteins and lipids. Although there is bioc......The lipid raft concept proposes that biological membranes have the potential to form functional domains based on a selective interaction between sphingolipids and sterols. These domains seem to be involved in signal transduction and vesicular sorting of proteins and lipids. Although...... there is biochemical evidence for lipid raft-dependent protein and lipid sorting in the yeast Saccharomyces cerevisiae, direct evidence for an interaction between yeast sphingolipids and the yeast sterol ergosterol, resulting in membrane domain formation, is lacking. Here we show that model membranes formed from yeast...... total lipid extracts possess an inherent self-organization potential resulting in Ld-Lo phase coexistence at physiologically relevant temperature. Analyses of lipid extracts from mutants defective in sphingolipid metabolism as well as reconstitution of purified yeast lipids in model membranes of defined...

INTRASURGICAL MICROSCOPE-INTEGRATED SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY-ASSISTED MEMBRANE PEELING.

Science.gov (United States)

Falkner-Radler, Christiane I; Glittenberg, Carl; Gabriel, Max; Binder, Susanne

2015-10-01

To evaluate microscope-integrated intrasurgical spectral domain optical coherence tomography during macular surgery in a prospective monocenter study. Before pars plana vitrectomy and before, during, and after membrane peeling, 512 × 128 macular cube scans were performed using a Carl Zeiss Meditec Cirrus high-definition OCT system adapted to the optical pathway of a Zeiss OPMI VISU 200 surgical microscope and compared with retinal staining. The study included 51 patients with epiretinal membranes, with 8 of those having additional lamellar macular holes, 11 patients with vitreomacular traction, and 8 patients with full-thickness macular holes. Intraoperative spectral domain optical coherence tomography allowed performing membrane peeling without using retinal dyes in 40% of cases (28 of 70 patients). No residual membranes were found in 94.3% of patients (66 of 70 patients) in intrasurgical spectral domain optical coherence tomography and subsequent (re)staining. In patients with vitreomacular traction, intrasurgical spectral domain optical coherence tomography scans facilitated decisions on the need for an intraocular tamponade after membrane peeling. Intraoperative spectral domain optical coherence tomography was comparable with retinal dyes in confirming success after membrane peeling. However, the visualization of flat membranes was better after staining.

Higher-order assemblies of BAR domain proteins for shaping membranes.

Science.gov (United States)

Suetsugu, Shiro

2016-06-01

Most cellular organelles contain lipid bilayer membranes. The earliest characterization of cellular organelles was performed by electron microscopy observation of such membranes. However, the precise mechanisms for shaping the membrane in particular subcellular organelles is poorly understood. Classically, the overall cellular shape, i.e. the shape of the plasma membrane, was thought to be governed by the reorganization of cytoskeletal components such as actin and microtubules. The plasma membrane contains various submicron structures such as clathrin-coated pits, caveolae, filopodia and lamellipodia. These subcellular structures are either invaginations or protrusions and are associated with the cytoskeleton. Therefore, it could be hypothesized that there are membrane-binding proteins that cooperates with cytoskeleton in shaping of plasma membrane organelles. Proteins with the Bin-Amphiphysin-Rvs (BAR) domain connect a variety of membrane shapes to actin filaments. The BAR domains themselves bend the membranes by their rigidity and then mold the membranes into tubules through their assembly as spiral polymers, which are thought to be involved in the various submicron structures. Membrane tubulation by polymeric assembly of the BAR domains is supposed to be regulated by binding proteins, binding lipids and the mechanical properties of the membrane. This review gives an overview of BAR protein assembly, describes the significance of the assembly and discusses how to study the assembly in the context of membrane and cellular morphology. The technical problems encountered in microscopic observation of BAR domain assembly are also discussed. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane[S

Science.gov (United States)

Carquin, Mélanie; Pollet, Hélène; Veiga-da-Cunha, Maria; Cominelli, Antoine; Van Der Smissen, Patrick; N’kuli, Francisca; Emonard, Hervé; Henriet, Patrick; Mizuno, Hideaki; Courtoy, Pierre J.; Tyteca, Donatienne

2014-01-01

We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin (SM) into living red blood cells (RBCs), partially spread onto coverslips, labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous SM, upon binding of a SM-specific nontoxic (NT) fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry [construct named His-mCherry-NT-lysenin (lysenin*)]. Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of 125I-lysenin* binding to erythrocytes upon SM depletion by SMase. The 125I-lysenin* binding isotherm indicated saturation at 3.5 × 106 molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled sub­micrometric domains on the plasma membrane of partially spread erythrocytes, colocalizing with inserted green BODIPY-SM, and abrogated by SMase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning, and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in RBCs gently suspended in 3D-gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous SM at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo. PMID:24826836

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

Directory of Open Access Journals (Sweden)

Antonin Lamazière

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

Formation of Kinetically Trapped Nanoscopic Unilamellar Vesicles from Metastable Nanodiscs

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Nieh, Mu-Ping [Univ. of Connecticut, Storrs, CT (United States). Inst. of Materials Science, Dept. of Chemical, Materials & Biomolecular Engineering; Dolinar, Paul [Univ. of Ottawa, ON (Canada); Kucerka, Norbert [National Research Council, Chalk River, ON (Canada). Chalk River Lab., Canadian Neutron Beam Centre; Comenius Univ., Bratislava (Slovakia). Dept. of Physical Chemistry of Drugs; Kline, Steven R. [National Inst. of Standards and Technology (NIST), Gaithersburg, MD (United States); Debeer-Schmitt, Lisa M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Science Division; Littrell, Kenneth C. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Science Division; Katsaras, John [National Research Council, Chalk River, ON (Canada). Chalk River Lab., Canadian Neutron Beam Centre; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Neutron Scattering Science Division; Brock Univ., St. Catharines, ON (Canada). Dept. of Physics; Univ. of Guelph, ON (Canada). Guelph-Waterloo Physics Inst.

2011-09-27

Zwitterionic long-chain lipids (e.g., dimyristoyl phosphatidylcholine, DMPC) spontaneously form onion-like, thermodynamically stable structures in aqueous solutions (commonly known as multilamellar vesicles, or MLVs). It has also been reported that the addition of zwitterionic short-chain (i.e., dihexanoyl phosphatidylcholine, DHPC) and charged long-chain (i.e., dimyristoyl phosphatidylglycerol, DMPG) lipids to zwitterionic long-chain lipid solutions results in the formation of unilamellar vesicles (ULVs). Here, we report a kinetic study on lipid mixtures composed of DMPC, DHPC, and DMPG. Two membrane charge densities (i.e., [DMPG]/[DMPC] = 0.01 and 0.001) and two solution salinities (i.e., [NaCl] = 0 and 0.2 M) are investigated. Upon dilution of the high-concentration samples at 50 °C, thermodynamically stable MLVs are formed, in the case of both weakly charged and high salinity solution mixtures, implying that the electrostatic interactions between bilayers are insufficient to cause MLVs to unbind. Importantly, in the case of these samples small angle neutron scattering (SANS) data show that, initially, nanodiscs (also known as bicelles) or bilayered ribbons form at low temperatures (i.e., 10 °C), but transform into uniform size, nanoscopic ULVs after incubation at 10 °C for 20 h, indicating that the nanodisc is a metastable structure. The instability of nanodiscs may be attributed to low membrane rigidity due to a reduced charge density and high salinity. Moreover, the uniform-sized ULVs persist even after being heated to 50 °C, where thermodynamically stable MLVs are observed. This result clearly demonstrates that these ULVs are kinetically trapped, and that the mechanical properties (e.g., bending rigidity) of 10 C nanodiscs favor the formation of nanoscopic ULVs over that of MLVs. From a practical point of view, this method of forming uniform-sized ULVs may lend itself to their mass production, thus making them economically feasible for medical

Formation of Kinetically Trapped Nanoscopic Unilamellar Vesicles from Metastable Nanodiscs

International Nuclear Information System (INIS)

Nieh, Mu-Ping; Dolinar, Paul; Kucerka, Norbert; Kline, Steven R.; Debeer-Schmitt, Lisa M.; Littrell, Ken; Katsaras, John

2011-01-01

Zwitterionic long-chain lipids (e.g., dimyristoyl phosphatidylcholine, DMPC) spontaneously form onion-like, thermodynamically stable structures in aqueous solutions (commonly known as multilamellar vesicles, or MLVs). It has also been reported that the addition of zwitterionic short-chain (i.e., dihexanoyl phosphatidylcholine, DHPC) and charged long-chain (i.e., dimyristoyl phosphatidylglycerol, DMPG) lipids to zwitterionic long-chain lipid solutions results in the formation of unilamellar vesicles (ULVs). Here, we report a kinetic study on lipid mixtures composed of DMPC, DHPC, and DMPG. Two membrane charge densities (i.e., (DMPG)/(DMPC) = 0.01 and 0.001) and two solution salinities (i.e., (NaCl) = 0 and 0.2 M) are investigated. Upon dilution of the high-concentration samples at 50 C, thermodynamically stable MLVs are formed, in the case of both weakly charged and high salinity solution mixtures, implying that the electrostatic interactions between bilayers are insufficient to cause MLVs to unbind. Importantly, in the case of these samples small angle neutron scattering (SANS) data show that, initially, nanodiscs (also known as bicelles) or bilayered ribbons form at low temperatures (i.e., 10 C), but transform into uniform size, nanoscopic ULVs after incubation at 10 C for 20 h, indicating that the nanodisc is a metastable structure. The instability of nanodiscs may be attributed to low membrane rigidity due to a reduced charge density and high salinity. Moreover, the uniform-sized ULVs persist even after being heated to 50 C, where thermodynamically stable MLVs are observed. This result clearly demonstrates that these ULVs are kinetically trapped, and that the mechanical properties (e.g., bending rigidity) of 10 C nanodiscs favor the formation of nanoscopic ULVs over that of MLVs. From a practical point of view, this method of forming uniform-sized ULVs may lend itself to their mass production, thus making them economically feasible for medical applications that

Effect of membrane microheterogeneity and domain size on fluorescence resonance energy transfer.

Science.gov (United States)

Towles, Kevin B; Brown, Angela C; Wrenn, Steven P; Dan, Nily

2007-07-15

Studies of multicomponent membranes suggest lateral inhomogeneity in the form of membrane domains, but the size of small (nanoscale) domains in situ cannot be determined with current techniques. In this article, we present a model that enables extraction of membrane domain size from time-resolved fluorescence resonance energy transfer (FRET) data. We expand upon a classic approach to the infinite phase separation limit and formulate a model that accounts for the presence of disklike domains of finite dimensions within a two-dimensional infinite planar bilayer. The model was tested against off-lattice Monte Carlo calculations of a model membrane in the liquid-disordered (l(d)) and liquid-ordered (l(o)) coexistence regime. Simulated domain size was varied from 5 to 50 nm, and two fluorophores, preferentially partitioning into opposite phases, were randomly mixed to obtain the simulated time-resolved FRET data. The Monte Carlo data show clear differences in the efficiency of energy transfer as a function of domain size. The model fit of the data yielded good agreement for the domain size, especially in cases where the domain diameter is membrane domains using time-resolved FRET.

Reorganization of plasma membrane lipid domains during conidial germination.

Science.gov (United States)

Santos, Filipa C; Fernandes, Andreia S; Antunes, Catarina A C; Moreira, Filipe P; Videira, Arnaldo; Marinho, H Susana; de Almeida, Rodrigo F M

2017-02-01

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth. Copyright © 2016 Elsevier B.V. All rights reserved.

Macroscopic domain formation in the platelet plasma membrane

DEFF Research Database (Denmark)

Bali, Rachna; Savino, Laura; Ramirez, Diego A.

2009-01-01

There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large d...

Distribution of macular xanthophylls between domains in a model of photoreceptor outer segment membranes.

Science.gov (United States)

Wisniewska, Anna; Subczynski, Witold K

2006-10-15

A model of photoreceptor outer segment (POS) membranes has been proposed, consisting of an equimolar ternary mixture of 1-palmitoyl-2-docosahexaenoylphosphatidylcholine/distearoylphosphatidylcholine/cholesterol. It was shown that, as in membranes made from the raft-forming mixture, in the model of POS membranes, two domains are formed: the raft domain (detergent resistant membranes, DRM), and the bulk domain (detergent soluble membranes, DSM). Saturation-recovery EPR discrimination by oxygen transport method also demonstrated the presence of two domains in this model system in situ at a wide range of temperatures (10-55 degrees C), showing additionally that neither lutein nor zeaxanthin at 1 mol% affect the formation of these domains. These membrane domains have been separated using cold Triton X-100 extraction from a model of POS membranes containing 1 mol% of either lutein or zeaxanthin. The results indicated that the macular xanthophylls lutein and zeaxanthin are substantially excluded from DRM and remain concentrated in DSM, a domain enriched in highly unsaturated docosahexaenoyl acid which is abundant in retina membranes. The concentration of xanthophylls in DRM and DSM calculated as the mol ratio of either xanthophyll to total lipid (phospholipid+cholesterol) was 0.0028 and 0.0391, respectively. Thus, xanthophylls are about 14 times more concentrated in DSM than in DRM. No significant difference in the distribution of lutein and zeaxanthin was found. The obtained results suggest that in POS membranes macular xanthophylls should also be concentrated in domains enriched in polyunsaturated chains.

Nanoscale Membrane Domain Formation Driven by Cholesterol

DEFF Research Database (Denmark)

Javanainen, Matti; Martinez-Seara, Hector; Vattulainen, Ilpo

2017-01-01

Biological membranes generate specific functions through compartmentalized regions such as cholesterol-enriched membrane nanodomains that host selected proteins. Despite the biological significance of nanodomains, details on their structure remain elusive. They cannot be observed via microscopic...... dipalmitoylphosphatidylcholine and cholesterol - the "minimal standard" for nanodomain formation. The simulations reveal how cholesterol drives the formation of fluid cholesterol-rich nanodomains hosting hexagonally packed cholesterol-poor lipid nanoclusters, both of which show registration between the membrane leaflets....... The complex nanodomain substructure forms when cholesterol positions itself in the domain boundary region. Here cholesterol can also readily flip-flop across the membrane. Most importantly, replacing cholesterol with a sterol characterized by a less asymmetric ring region impairs the emergence of nanodomains...

Imaging lipid domains in cell membranes: the advent of super-resolution fluorescence microscopy

Directory of Open Access Journals (Sweden)

Dylan Myers Owen

2013-12-01

Full Text Available The lipid bilayer of model membranes, liposomes reconstituted from cell lipids, and plasma membrane vesicles and spheres can separate into two distinct liquid phases to yield lipid domains with liquid-ordered and liquid-disordered properties. These observations are the basis of the lipid raft hypothesis that postulates the existence of cholesterol-enriched ordered-phase lipid domains in cell membranes that could regulate protein mobility, localization and interaction. Here we review the evidence that nano-scaled lipid complexes and meso-scaled lipid domains exist in cell membranes and how new fluorescence microscopy techniques that overcome the diffraction limit provide new insights into lipid organization in cell membranes.

Structure and hydration of membranes embedded with voltage-sensing domains.

Science.gov (United States)

Krepkiy, Dmitriy; Mihailescu, Mihaela; Freites, J Alfredo; Schow, Eric V; Worcester, David L; Gawrisch, Klaus; Tobias, Douglas J; White, Stephen H; Swartz, Kenton J

2009-11-26

Despite the growing number of atomic-resolution membrane protein structures, direct structural information about proteins in their native membrane environment is scarce. This problem is particularly relevant in the case of the highly charged S1-S4 voltage-sensing domains responsible for nerve impulses, where interactions with the lipid bilayer are critical for the function of voltage-activated ion channels. Here we use neutron diffraction, solid-state nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations to investigate the structure and hydration of bilayer membranes containing S1-S4 voltage-sensing domains. Our results show that voltage sensors adopt transmembrane orientations and cause a modest reshaping of the surrounding lipid bilayer, and that water molecules intimately interact with the protein within the membrane. These structural findings indicate that voltage sensors have evolved to interact with the lipid membrane while keeping energetic and structural perturbations to a minimum, and that water penetrates the membrane, to hydrate charged residues and shape the transmembrane electric field.

Protein shape and crowding drive domain formation and curvature in biological membranes

NARCIS (Netherlands)

Frese, R.N.; Pamies, Josep C.; Olsen, John D.; Bahatyrova, S.; van der Weij-de Wit, Chantal D.; Aartsma, Thijs J.; Otto, Cornelis; Hunter, C. Neil; Frenkel, Daan; van Grondelle, Rienk

2007-01-01

Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization

Functional polymers as nanoscopic building blocks

International Nuclear Information System (INIS)

Hernandez-Lopez, J.L.; Bauer, R.E.; Chang, W.-S.; Glasser, G.; Grebel-Koehler, D.; Klapper, M.; Kreiter, M.; Leclaire, J.; Majoral, J.-P.; Mittler, S.; Muellen, K.; Vasilev, K.; Weil, T.; Wu, J.; Zhu, T.; Knoll, W.

2003-01-01

Polyphenylene dendrimers are introduced as polymeric building blocks--with a strictly monodisperse particle size distribution within the nanometer range--for the construction of nanostructured materials and devices. The possibility for the introduction of different functionalities in the core, the scaffold or the periphery of the dendrimers offer their use as interesting modules for photonic, electronic or bioactive structures and supramolecular functional assemblies. Thus, dendrimers complement the available set of nanoscopic building blocks made from metals, e.g., Au nanoclusters and semiconductors, e.g., luminescent quantum dots. In a first set of experiments, we describe the fabrication of multilayer architectures using dendrimers with chargeable groups at the surface. This way, the polyelectrolyte deposition technique can be applied for the construction of hybrid layered assemblies with a control of the internal supramolecular structure at the nanometer level. Surface plasmon field-enhanced fluorescence spectroscopy is used to monitor the luminescent properties of dendrimers with a phthalocyanine core integrated into such a multilayer assembly. AFM and SEM micrographs demonstrate the use of surface-functionalized dendrimers (exposing sulfur groups at the periphery) in combination with Au nanoparticles for the controlled assembly of hybrid aggregates as nanoscopic functional devices

Membrane Localization is Critical for Activation of the PICK1 BAR Domain

Science.gov (United States)

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

2013-01-01

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

Recent progress on lipid lateral heterogeneity in plasma membranes: from rafts to submicrometric domains

Science.gov (United States)

Carquin, Mélanie; D'Auria, Ludovic; Pollet, Hélène; Bongarzone, Ernesto R.; Tyteca, Donatienne

2016-01-01

The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicholson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decade, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (> min vs sec) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryotes to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution. PMID:26738447

Accumulation of raft lipids in T-cell plasma membrane domains engaged in TCR signalling

DEFF Research Database (Denmark)

Zech, Tobias; Ejsing, Christer S.; Gaus, Katharina

2009-01-01

Activating stimuli for T lymphocytes are transmitted through plasma membrane domains that form at T-cell antigen receptor (TCR) signalling foci. Here, we determined the molecular lipid composition of immunoisolated TCR activation domains. We observed that they accumulate cholesterol, sphingomyelin...... and saturated phosphatidylcholine species as compared with control plasma membrane fragments. This provides, for the first time, direct evidence that TCR activation domains comprise a distinct molecular lipid composition reminiscent of liquid-ordered raft phases in model membranes. Interestingly, TCR activation...... domains were also enriched in plasmenyl phosphatidylethanolamine and phosphatidylserine. Modulating the T-cell lipidome with polyunsaturated fatty acids impaired the plasma membrane condensation at TCR signalling foci and resulted in a perturbed molecular lipid composition. These results correlate...

Contribution of Adsorbed Protein Films to Nanoscopic Vibrations Exhibited by Bacteria Adhering through Ligand-Receptor Bonds

NARCIS (Netherlands)

Song, Lei; Sjollema, Jelmer; Norde, Willem; Busscher, Henk J.; van der Mei, Henny C.

2015-01-01

Bacteria adhering to surfaces exhibit nanoscopic vibrations that depend on the viscoelasticity of the bond. The quantification of the nanoscopic vibrations of bacteria adhering to surfaces provides new opportunities to better understand the properties of the bond through which bacteria adhere and

Magnetic susceptibility in the edged topological disordered nanoscopic cylinder

International Nuclear Information System (INIS)

Faizabadi, Edris; Omidi, Mahboubeh

2011-01-01

The effects of edged topological disorder on magnetic susceptibility are investigated in a nanoscopic cylinder threaded by a magnetic flux. Persistent current versus even or odd number of electrons shows different signs in ordered and disordered cylinders and also in short or long ones. In addition, temperature-averaged susceptibility has only diamagnetic signs in strong regimes and it is associated with paramagnetic signs in ordered and weak disordered ones. Besides, in an edged topological disordered cylinder, the temperature-averaged susceptibility decreases by raising the temperature somewhat and then increasing initiates and finally at high temperature tends to zero as the ordered one. - Research highlights: → Magnetic susceptibility in one-dimensional topological disordered quantum ring. → Edged topological disorder effect on magnetic susceptibility in nanoscopic cylinder. → Edged topological disorder effect on temperature-averaged susceptibility in cylinder.

Visualizing Nanoscopic Topography and Patterns in Freely Standing Thin Films

Science.gov (United States)

Yilixiati, Subinuer; Zhang, Yiran; Pearsall, Collin; Sharma, Vivek

Thin liquid films containing micelles, nanoparticles, polyelectrolyte-surfactant complexes and smectic liquid crystals undergo thinning in a discontinuous, step-wise fashion. The discontinuous jumps in thickness are often characterized by quantifying changes in the intensity of reflected monochromatic light, modulated by thin film interference from a region of interest. Stratifying thin films exhibit a mosaic pattern in reflected white light microscopy, attributed to the coexistence of domains with various thicknesses, separated by steps. Using Interferometry Digital Imaging Optical Microscopy (IDIOM) protocols developed in the course of this study, we spatially resolve for the first time, the landscape of stratifying freestanding thin films. In particular, for thin films containing micelles of sodium dodecyl sulfate (SDS), discontinuous, thickness transitions with concentration-dependent steps of 5-25 nm are visualized and analyzed using IDIOM protocols. We distinguish nanoscopic rims, mesas and craters and show that the non-flat features are sculpted by oscillatory, periodic, supramolecular structural forces that arise in confined fluids

BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

DEFF Research Database (Denmark)

Madsen, Kenneth Lindegaard; Bhatia, V K; Gether, U

2010-01-01

/ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can...

Trp[superscript 2313]-His[superscript 2315] of Factor VIII C2 Domain Is Involved in Membrane Binding Structure of a Complex Between the C[subscript 2] Domain and an Inhibitor of Membrane Binding

Energy Technology Data Exchange (ETDEWEB)

Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong (Harvard-Med); (UAH); (Maastricht); (Chinese Aca. Sci.)

2010-11-03

Factor VIII (FVIII) plays a critical role in blood coagulation by forming the tenase complex with factor IXa and calcium ions on a membrane surface containing negatively charged phospholipids. The tenase complex activates factor X during blood coagulation. The carboxyl-terminal C2 domain of FVIII is the main membrane-binding and von Willebrand factor-binding region of the protein. Mutations of FVIII cause hemophilia A, whereas elevation of FVIII activity is a risk factor for thromboembolic diseases. The C2 domain-membrane interaction has been proposed as a target of intervention for regulation of blood coagulation. A number of molecules that interrupt FVIII or factor V (FV) binding to cell membranes have been identified through high throughput screening or structure-based design. We report crystal structures of the FVIII C2 domain under three new crystallization conditions, and a high resolution (1.15 {angstrom}) crystal structure of the FVIII C2 domain bound to a small molecular inhibitor. The latter structure shows that the inhibitor binds to the surface of an exposed {beta}-strand of the C2 domain, Trp{sup 2313}-His{sup 2315}. This result indicates that the Trp{sup 2313}-His{sup 2315} segment is an important constituent of the membrane-binding motif and provides a model to understand the molecular mechanism of the C2 domain membrane interaction.

Different methods of membrane domains isolation result in similar 2-D distribution patterns of membrane domain proteins

Czech Academy of Sciences Publication Activity Database

Matoušek, Petr; Hodný, Zdeněk; Švandová, I.; Svoboda, Petr

2003-01-01

Roč. 81, č. 6 (2003), s. 365-372 ISSN 0829-8211 R&D Projects: GA MŠk LN00A026 Grant - others:Wellcome Trust(GB) xx Institutional research plan: CEZ:AV0Z5011922; CEZ:MSM 113100003; CEZ:AV0Z5039906 Keywords : membrane domain * G protein * two-dimensional electrophoresis * GPI-ancored proteins Subject RIV: CE - Biochemistry Impact factor: 2.456, year: 2003

Assembly of the membrane domain of ATP synthase in human mitochondria.

Science.gov (United States)

He, Jiuya; Ford, Holly C; Carroll, Joe; Douglas, Corsten; Gonzales, Evvia; Ding, Shujing; Fearnley, Ian M; Walker, John E

2018-03-20

The ATP synthase in human mitochondria is a membrane-bound assembly of 29 proteins of 18 kinds. All but two membrane components are encoded in nuclear genes, synthesized on cytoplasmic ribosomes, and imported into the matrix of the organelle, where they are assembled into the complex with ATP6 and ATP8, the products of overlapping genes in mitochondrial DNA. Disruption of individual human genes for the nuclear-encoded subunits in the membrane portion of the enzyme leads to the formation of intermediate vestigial ATPase complexes that provide a description of the pathway of assembly of the membrane domain. The key intermediate complex consists of the F 1 -c 8 complex inhibited by the ATPase inhibitor protein IF 1 and attached to the peripheral stalk, with subunits e, f, and g associated with the membrane domain of the peripheral stalk. This intermediate provides the template for insertion of ATP6 and ATP8, which are synthesized on mitochondrial ribosomes. Their association with the complex is stabilized by addition of the 6.8 proteolipid, and the complex is coupled to ATP synthesis at this point. A structure of the dimeric yeast F o membrane domain is consistent with this model of assembly. The human 6.8 proteolipid (yeast j subunit) locks ATP6 and ATP8 into the membrane assembly, and the monomeric complexes then dimerize via interactions between ATP6 subunits and between 6.8 proteolipids (j subunits). The dimers are linked together back-to-face by DAPIT (diabetes-associated protein in insulin-sensitive tissue; yeast subunit k), forming long oligomers along the edges of the cristae.

MPP1 directly interacts with flotillins in erythrocyte membrane - Possible mechanism of raft domain formation.

Science.gov (United States)

Biernatowska, Agnieszka; Augoff, Katarzyna; Podkalicka, Joanna; Tabaczar, Sabina; Gajdzik-Nowak, Weronika; Czogalla, Aleksander; Sikorski, Aleksander F

2017-11-01

Flotillins are prominent, oligomeric protein components of erythrocyte (RBC) membrane raft domains and are considered to play an important structural role in lateral organization of the plasma membrane. In our previous work on erythroid membranes and giant plasma membrane vesicles (GPMVs) derived from them we have shown that formation of functional domains (resting state rafts) depends on the presence of membrane palmitoylated protein 1 (MPP1/p55), pointing to its new physiological role. Exploration of the molecular mechanism of MPP1 function in organizing membrane domains described here, through searching for its molecular partners in RBC membrane by using different methods, led to the identification of the raft-marker proteins, flotillin 1 and flotillin 2, as hitherto unreported direct MPP1 binding-partners in the RBC membrane. These proteins are found in high molecular-weight complexes in native RBC membrane and, significantly, their presence was shown to be separate from the well-known protein 4.1-dependent interactions of MPP1 with membrane proteins. Furthermore, FLIM analysis revealed that loss of the endogenous MPP1-flotillins interactions resulted in significant changes in RBC membrane-fluidity, emphasizing the physiological importance of such interactions in vivo. Therefore, our data establish a new perspective on the role of MPP1 in erythroid cells and suggests that direct MPP1-flotillins interactions could be the major driving-force behind the formation of raft domains in RBC. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Tracking cholesterol/sphingomyelin-rich membrane domains with the ostreolysin A-mCherry protein.

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Matej Skočaj

Full Text Available Ostreolysin A (OlyA is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II-Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1-positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.

Multifunctionalities driven by ferroic domains

Science.gov (United States)

Yang, J. C.; Huang, Y. L.; He, Q.; Chu, Y. H.

2014-08-01

Considerable attention has been paid to ferroic systems in pursuit of advanced applications in past decades. Most recently, the emergence and development of multiferroics, which exhibit the coexistence of different ferroic natures, has offered a new route to create functionalities in the system. In this manuscript, we step from domain engineering to explore a roadmap for discovering intriguing phenomena and multifunctionalities driven by periodic domain patters. As-grown periodic domains, offering exotic order parameters, periodic local perturbations and the capability of tailoring local spin, charge, orbital and lattice degrees of freedom, are introduced as modeling templates for fundamental studies and novel applications. We discuss related significant findings on ferroic domain, nanoscopic domain walls, and conjunct heterostructures based on the well-organized domain patterns, and end with future prospects and challenges in the field.

Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

Energy Technology Data Exchange (ETDEWEB)

Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A. (UPENN-MED)

2011-09-28

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

Membrane localization is critical for activation of the PICK1 BAR domain

DEFF Research Database (Denmark)

Madsen, Kenneth L; Eriksen, Jacob; Milan-Lobo, Laura

2008-01-01

The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood....

Confining Domains Lead to Reaction Bursts: Reaction Kinetics in the Plasma Membrane

Science.gov (United States)

Kalay, Ziya; Fujiwara, Takahiro K.; Kusumi, Akihiro

2012-01-01

Confinement of molecules in specific small volumes and areas within a cell is likely to be a general strategy that is developed during evolution for regulating the interactions and functions of biomolecules. The cellular plasma membrane, which is the outermost membrane that surrounds the entire cell, was considered to be a continuous two-dimensional liquid, but it is becoming clear that it consists of numerous nano-meso-scale domains with various lifetimes, such as raft domains and cytoskeleton-induced compartments, and membrane molecules are dynamically trapped in these domains. In this article, we give a theoretical account on the effects of molecular confinement on reversible bimolecular reactions in a partitioned surface such as the plasma membrane. By performing simulations based on a lattice-based model of diffusion and reaction, we found that in the presence of membrane partitioning, bimolecular reactions that occur in each compartment proceed in bursts during which the reaction rate is sharply and briefly increased even though the asymptotic reaction rate remains the same. We characterized the time between reaction bursts and the burst amplitude as a function of the model parameters, and discussed the biological significance of the reaction bursts in the presence of strong inhibitor activity. PMID:22479350

Confining domains lead to reaction bursts: reaction kinetics in the plasma membrane.

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Ziya Kalay

Full Text Available Confinement of molecules in specific small volumes and areas within a cell is likely to be a general strategy that is developed during evolution for regulating the interactions and functions of biomolecules. The cellular plasma membrane, which is the outermost membrane that surrounds the entire cell, was considered to be a continuous two-dimensional liquid, but it is becoming clear that it consists of numerous nano-meso-scale domains with various lifetimes, such as raft domains and cytoskeleton-induced compartments, and membrane molecules are dynamically trapped in these domains. In this article, we give a theoretical account on the effects of molecular confinement on reversible bimolecular reactions in a partitioned surface such as the plasma membrane. By performing simulations based on a lattice-based model of diffusion and reaction, we found that in the presence of membrane partitioning, bimolecular reactions that occur in each compartment proceed in bursts during which the reaction rate is sharply and briefly increased even though the asymptotic reaction rate remains the same. We characterized the time between reaction bursts and the burst amplitude as a function of the model parameters, and discussed the biological significance of the reaction bursts in the presence of strong inhibitor activity.

Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis.

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Zhang, Hongjie; Abraham, Nessy; Khan, Liakot A; Hall, David H; Fleming, John T; Göbel, Verena

2011-09-18

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions. Follow-on targeted lipid-biosynthesis pathway screens and functional genetic assays were designed to identify a putative single causative lipid species. They demonstrate that fatty-acid biosynthesis affects polarity through sphingolipid synthesis, and reveal ceramide glucosyltransferases (CGTs) as end-point biosynthetic enzymes in this pathway. Our findings identify glycosphingolipids, CGT products and obligate membrane lipids, as critical determinants of in vivo polarity and indicate that they sort new components to the expanding apical membrane.

Membrane Localization is Critical for Activation of the PICK1 BAR Domain

OpenAIRE

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

2008-01-01

The PSD-95/Discs-large/ZO-1 homology (PDZ) domain protein, protein interacting with C kinase 1 (PICK1) contains a C-terminal Bin/amphiphysin/Rvs (BAR) domain mediating recognition of curved membranes; however, the molecular mechanisms controlling the activity of this domain are poorly understood. In agreement with negative regulation of the BAR domain by the N-terminal PDZ domain, PICK1 distributed evenly in the cytoplasm, whereas truncation of the PDZ domain caused BAR domain-dependent redis...

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

DEFF Research Database (Denmark)

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

2008-01-01

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

Protein sorting by lipid phase-like domains supports emergent signaling function in B lymphocyte plasma membranes.

Science.gov (United States)

Stone, Matthew B; Shelby, Sarah A; Núñez, Marcos F; Wisser, Kathleen; Veatch, Sarah L

2017-02-01

Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.

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

Science.gov (United States)

Ronzone, Erik; Paumet, Fabienne

2013-01-01

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

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.

Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

Science.gov (United States)

Takatori, Sho; Tatematsu, Tsuyako; Cheng, Jinglei; Matsumoto, Jun; Akano, Takuya; Fujimoto, Toyoshi

2016-02-01

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Saturation recovery EPR spin-labeling method for quantification of lipids in biological membrane domains.

Science.gov (United States)

Mainali, Laxman; Camenisch, Theodore G; Hyde, James S; Subczynski, Witold K

2017-12-01

The presence of integral membrane proteins induces the formation of distinct domains in the lipid bilayer portion of biological membranes. Qualitative application of both continuous wave (CW) and saturation recovery (SR) electron paramagnetic resonance (EPR) spin-labeling methods allowed discrimination of the bulk, boundary, and trapped lipid domains. A recently developed method, which is based on the CW EPR spectra of phospholipid (PL) and cholesterol (Chol) analog spin labels, allows evaluation of the relative amount of PLs (% of total PLs) in the boundary plus trapped lipid domain and the relative amount of Chol (% of total Chol) in the trapped lipid domain [ M. Raguz, L. Mainali, W. J. O'Brien, and W. K. Subczynski (2015), Exp. Eye Res., 140:179-186 ]. Here, a new method is presented that, based on SR EPR spin-labeling, allows quantitative evaluation of the relative amounts of PLs and Chol in the trapped lipid domain of intact membranes. This new method complements the existing one, allowing acquisition of more detailed information about the distribution of lipids between domains in intact membranes. The methodological transition of the SR EPR spin-labeling approach from qualitative to quantitative is demonstrated. The abilities of this method are illustrated for intact cortical and nuclear fiber cell plasma membranes from porcine eye lenses. Statistical analysis (Student's t -test) of the data allowed determination of the separations of mean values above which differences can be treated as statistically significant ( P ≤ 0.05) and can be attributed to sources other than preparation/technique.

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

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Alexey S. Ladokhin

2013-08-01

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

The dynamic interplay of plasma membrane domains and cortical microtubules in secondary cell wall patterning

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Yoshihisa eOda

2013-12-01

Full Text Available Patterning of the cellulosic cell wall underlies the shape and function of plant cells. The cortical microtubule array plays a central role in the regulation of cell wall patterns. However, the regulatory mechanisms by which secondary cell wall patterns are established through cortical microtubules remain to be fully determined. Our recent study in xylem vessel cells revealed that a mutual inhibitory interaction between cortical microtubules and distinct plasma membrane domains leads to distinctive patterning in secondary cell walls. Our research revealed that the recycling of active and inactive ROP proteins by a specific GAP and GEF pair establishes distinct de novo plasma membrane domains. Active ROP recruits a plant-specific microtubule-associated protein, MIDD1, which mediates the mutual interaction between cortical microtubules and plasma membrane domains. In this mini review, we summarize recent research regarding secondary wall patterning, with a focus on the emerging interplay between plasma membrane domains and cortical microtubules through MIDD1 and ROP.

Building a patchwork - The yeast plasma membrane as model to study lateral domain formation.

Science.gov (United States)

Schuberth, Christian; Wedlich-Söldner, Roland

2015-04-01

The plasma membrane (PM) has to fulfill a wide range of biological functions including selective uptake of substances, signal transduction and modulation of cell polarity and cell shape. To allow efficient regulation of these processes many resident proteins and lipids of the PM are laterally segregated into different functional domains. A particularly striking example of lateral segregation has been described for the budding yeast PM, where integral membrane proteins as well as lipids exhibit very slow translational mobility and form a patchwork of many overlapping micron-sized domains. Here we discuss the molecular and physical mechanisms contributing to the formation of a multi-domain membrane and review our current understanding of yeast PM organization. Many of the fundamental principles underlying membrane self-assembly and organization identified in yeast are expected to equally hold true in other organisms, even for the more transient and elusive organization of the PM in mammalian cells. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling. Copyright © 2014 Elsevier B.V. All rights reserved.

Regulation of adhesion behavior of murine macrophage using supported lipid membranes displaying tunable mannose domains

International Nuclear Information System (INIS)

Kaindl, T; Oelke, J; Kaufmann, S; Tanaka, M; Pasc, A; Konovalov, O V; Funari, S S; Engel, U; Wixforth, A

2010-01-01

Highly uniform, strongly correlated domains of synthetically designed lipids can be incorporated into supported lipid membranes. The systematic characterization of membranes displaying a variety of domains revealed that the equilibrium size of domains significantly depends on the length of fluorocarbon chains, which can be quantitatively interpreted within the framework of an equivalent dipole model. A mono-dispersive, narrow size distribution of the domains enables us to treat the inter-domain correlations as two-dimensional colloidal crystallization and calculate the potentials of mean force. The obtained results demonstrated that both size and inter-domain correlation can precisely be controlled by the molecular structures. By coupling α-D-mannose to lipid head groups, we studied the adhesion behavior of the murine macrophage (J774A.1) on supported membranes. Specific adhesion and spreading of macrophages showed a clear dependence on the density of functional lipids. The obtained results suggest that such synthetic lipid domains can be used as a defined platform to study how cells sense the size and distribution of functional molecules during adhesion and spreading.

Spatial cluster analysis of nanoscopically mapped serotonin receptors for classification of fixed brain tissue

Science.gov (United States)

Sams, Michael; Silye, Rene; Göhring, Janett; Muresan, Leila; Schilcher, Kurt; Jacak, Jaroslaw

2014-01-01

We present a cluster spatial analysis method using nanoscopic dSTORM images to determine changes in protein cluster distributions within brain tissue. Such methods are suitable to investigate human brain tissue and will help to achieve a deeper understanding of brain disease along with aiding drug development. Human brain tissue samples are usually treated postmortem via standard fixation protocols, which are established in clinical laboratories. Therefore, our localization microscopy-based method was adapted to characterize protein density and protein cluster localization in samples fixed using different protocols followed by common fluorescent immunohistochemistry techniques. The localization microscopy allows nanoscopic mapping of serotonin 5-HT1A receptor groups within a two-dimensional image of a brain tissue slice. These nanoscopically mapped proteins can be confined to clusters by applying the proposed statistical spatial analysis. Selected features of such clusters were subsequently used to characterize and classify the tissue. Samples were obtained from different types of patients, fixed with different preparation methods, and finally stored in a human tissue bank. To verify the proposed method, samples of a cryopreserved healthy brain have been compared with epitope-retrieved and paraffin-fixed tissues. Furthermore, samples of healthy brain tissues were compared with data obtained from patients suffering from mental illnesses (e.g., major depressive disorder). Our work demonstrates the applicability of localization microscopy and image analysis methods for comparison and classification of human brain tissues at a nanoscopic level. Furthermore, the presented workflow marks a unique technological advance in the characterization of protein distributions in brain tissue sections.

Nanoscopic Manipulation and Imaging of Liquid Crystals

Energy Technology Data Exchange (ETDEWEB)

Rosenblatt, Charles S. [Case Western Reserve Univ., Cleveland, OH (United States)

2014-02-04

This is the final project report. The project’s goals centered on nanoscopic imaging and control of liquid crystals and surfaces. We developed and refined techniques to control liquid crystal orientation at surfaces with resolution as small as 25 nm, we developed an optical imaging technique that we call Optical Nanotomography that allows us to obtain images inside liquid crystal films with resolution of 60 x 60 x 1 nm, and we opened new thrust areas related to chirality and to liquid crystal/colloid composites.

Effect of Dialkyl Ammonium Cationic Surfactants on the Microfluidity of Membranes Containing Raft Domains.

Science.gov (United States)

Uyama, Makoto; Inoue, Kaori; Kinoshita, Koichi; Miyahara, Reiji; Yokoyama, Hirokazu; Nakano, Minoru

2018-01-01

It has been reported that a lot of receptors localize in lipid raft domains and that the microfluidity of these domains regulates the activation of these receptors. In this study, we focused on the lipid raft and in order to evaluate the physicochemical effects of surfactants on microfluidity of lipid membranes, we used liposomes comprising of egg-yolk L-α-phosphatidylcholine, egg-yolk sphingomyelin, and cholesterol as a model of cell membranes containing raft domains. The microfluidity of the domains was characterized by fluorescence spectrometry using 1,6-diphenyl-1,3,5-hexatriene and 2-dimethylamino-6-lauroylnaphthalene. Among several surfactants, dialkylammonium-type cationic surfactants most efficiently increased the microfluidity. It is therefore concluded that (1) the electrostatic interaction between the cationic surfactant and eggPC/eggSM/cholesterol liposome could be important, (2) surfactants with alkyl chains more effectively inserted into membranes than those with acyl chains, and (3) cationic surfactants with lower T m values have a greater ability to increase the fluidity.

Membrane re-modelling by BAR domain superfamily proteins via molecular and non-molecular factors.

Science.gov (United States)

Nishimura, Tamako; Morone, Nobuhiro; Suetsugu, Shiro

2018-04-17

Lipid membranes are structural components of cell surfaces and intracellular organelles. Alterations in lipid membrane shape are accompanied by numerous cellular functions, including endocytosis, intracellular transport, and cell migration. Proteins containing Bin-Amphiphysin-Rvs (BAR) domains (BAR proteins) are unique, because their structures correspond to the membrane curvature, that is, the shape of the lipid membrane. BAR proteins present at high concentration determine the shape of the membrane, because BAR domain oligomers function as scaffolds that mould the membrane. BAR proteins co-operate with various molecular and non-molecular factors. The molecular factors include cytoskeletal proteins such as the regulators of actin filaments and the membrane scission protein dynamin. Lipid composition, including saturated or unsaturated fatty acid tails of phospholipids, also affects the ability of BAR proteins to mould the membrane. Non-molecular factors include the external physical forces applied to the membrane, such as tension and friction. In this mini-review, we will discuss how the BAR proteins orchestrate membrane dynamics together with various molecular and non-molecular factors. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Correlation between the ripple phase and stripe domains in membranes.

Science.gov (United States)

Bernchou, Uffe; Midtiby, Henrik; Ipsen, John Hjort; Simonsen, Adam Cohen

2011-12-01

We investigate the relationship between stripe domains and the ripple phase in membranes. These have previously been observed separately without being linked explicitly. Past results have demonstrated that solid and ripple phases exhibit rich textural patterns related to the orientational order of tilted lipids and the orientation of ripple corrugations. Here we reveal a highly complex network pattern of ripple and solid domains in DLPC, DPPC bilayers with structures covering length scales from 10 nm to 100 μm. Using spincoated double supported membranes we investigate domains by correlated AFM and fluorescence microscopy. Cooling experiments demonstrate the mode of nucleation and growth of stripe domains enriched in the fluorescent probe. Concurrent AFM imaging reveals that these stripe domains have a one-to-one correspondence with a rippled morphology running parallel to the stripe direction. Both thin and thick stripe domains are observed having ripple periods of 13.5±0.2 nm and 27.4±0.6 nm respectively. These are equivalent to previously observed asymmetric/equilibrium and symmetric/metastable ripple phases, respectively. Thin stripes grow from small solid domains and grow predominantly in length with a speed of ~3 times that of the thick stripes. Thick stripes grow by templating on the sides of thinner stripes or can emerge directly from the fluid phase. Bending and branching angles of stripes are in accordance with an underlying six fold lattice. We discuss mechanisms for the nucleation and growth of ripples and discuss a generic phase diagram that may partly rationalize the coexistence of metastable and stable phases. Copyright © 2011 Elsevier B.V. All rights reserved.

Membrane binding properties of EBV gp110 C-terminal domain; evidences for structural transition in the membrane environment

International Nuclear Information System (INIS)

Park, Sung Jean; Seo, Min-Duk; Lee, Suk Kyeong; Lee, Bong Jin

2008-01-01

Gp110 of Epstein-Barr virus (EBV) mainly localizes on nuclear/ER membranes and plays a role in the assembly of EBV nucleocapsid. The C-terminal tail domain (gp110 CTD) is essential for the function of gp110 and the nuclear/ER membranes localization of gp110 is ruled by its C-terminal unique nuclear localization signal (NLS), consecutive four arginines. In the present study, the structural properties of gp110 CTD in membrane mimics were investigated using CD, size-exclusion chromatography, and NMR, to elucidate the effect of membrane environment on the structural transition and to compare the structural feature of the protein in the solution state with that of the membrane-bound form. CD and NMR analysis showed that gp110 CTD in a buffer solution appears to adopt a stable folding intermediate which lacks compactness, and a highly helical structure is formed only in membrane environments. The helical content of gp110 CTD was significantly affected by the negative charge as well as the size of membrane mimics. Based on the elution profiles of the size-exclusion chromatography, we found that gp110 CTD intrinsically forms a trimer, revealing that a trimerization region may exist in the C-terminal domain of gp110 like the ectodomain of gp110. The mutation of NLS (RRRR) to RTTR does not affect the overall structure of gp110 CTD in membrane mimics, while the helical propensity in a buffer solution was slightly different between the wild-type and the mutant proteins. This result suggests that not only the helicity induced in membrane environment but also the local structure around NLS may be related to trafficking to the nuclear membrane. More detailed structural difference between the wild-type and the mutant in membrane environment was examined using synthetic two peptides including the wild-type NLS and the mutant NLS

A Coincidence Detection Mechanism Controls PX-BAR Domain-Mediated Endocytic Membrane Remodeling via an Allosteric Structural Switch.

Science.gov (United States)

Lo, Wen-Ting; Vujičić Žagar, Andreja; Gerth, Fabian; Lehmann, Martin; Puchkov, Dymtro; Krylova, Oxana; Freund, Christian; Scapozza, Leonardo; Vadas, Oscar; Haucke, Volker

2017-11-20

Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P 2 ) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P 2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Mesoscale organization of domains in the plasma membrane - beyond the lipid raft.

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Lu, Stella M; Fairn, Gregory D

2018-04-01

The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid-protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the "lipid raft" concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms "membrane raft" or "membrane nanodomain" are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.

Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking.

Science.gov (United States)

Lee, Jennifer; Ding, ShuJing; Walpole, Thomas B; Holding, Andrew N; Montgomery, Martin G; Fearnley, Ian M; Walker, John E

2015-05-22

The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Organization of Subunits in the Membrane Domain of the Bovine F-ATPase Revealed by Covalent Cross-linking*

Science.gov (United States)

Lee, Jennifer; Ding, ShuJing; Walpole, Thomas B.; Holding, Andrew N.; Montgomery, Martin G.; Fearnley, Ian M.; Walker, John E.

2015-01-01

The F-ATPase in bovine mitochondria is a membrane-bound complex of about 30 subunits of 18 different kinds. Currently, ∼85% of its structure is known. The enzyme has a membrane extrinsic catalytic domain, and a membrane intrinsic domain where the turning of the enzyme's rotor is generated from the transmembrane proton-motive force. The domains are linked by central and peripheral stalks. The central stalk and a hydrophobic ring of c-subunits in the membrane domain constitute the enzyme's rotor. The external surface of the catalytic domain and membrane subunit a are linked by the peripheral stalk, holding them static relative to the rotor. The membrane domain contains six additional subunits named ATP8, e, f, g, DAPIT (diabetes-associated protein in insulin-sensitive tissues), and 6.8PL (6.8-kDa proteolipid), each with a single predicted transmembrane α-helix, but their orientation and topography are unknown. Mutations in ATP8 uncouple the enzyme and interfere with its assembly, but its roles and the roles of the other five subunits are largely unknown. We have reacted accessible amino groups in the enzyme with bifunctional cross-linking agents and identified the linked residues. Cross-links involving the supernumerary subunits, where the structures are not known, show that the C terminus of ATP8 extends ∼70 Å from the membrane into the peripheral stalk and that the N termini of the other supernumerary subunits are on the same side of the membrane, probably in the mitochondrial matrix. These experiments contribute significantly toward building up a complete structural picture of the F-ATPase. PMID:25851905

Multiscale Simulations Suggest a Mechanism for the Association of the Dok7 PH Domain with PIP-Containing Membranes.

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Amanda Buyan

2016-07-01

Full Text Available Dok7 is a peripheral membrane protein that is associated with the MuSK receptor tyrosine kinase. Formation of the Dok7/MuSK/membrane complex is required for the activation of MuSK. This is a key step in the complex exchange of signals between neuron and muscle, which lead to neuromuscular junction formation, dysfunction of which is associated with congenital myasthenic syndromes. The Dok7 structure consists of a Pleckstrin Homology (PH domain and a Phosphotyrosine Binding (PTB domain. The mechanism of the Dok7 association with the membrane remains largely unknown. Using multi-scale molecular dynamics simulations we have explored the formation of the Dok7 PH/membrane complex. Our simulations indicate that the PH domain of Dok7 associates with membranes containing phosphatidylinositol phosphates (PIPs via interactions of the β1/β2, β3/β4, and β5/β6 loops, which together form a positively charged surface on the PH domain and interact with the negatively charged headgroups of PIP molecules. The initial encounter of the Dok7 PH domain is followed by formation of additional interactions with the lipid bilayer, and especially with PIP molecules, which stabilizes the Dok7 PH/membrane complex. We have quantified the binding of the PH domain to the model bilayers by calculating a density landscape for protein/membrane interactions. Detailed analysis of the PH/PIP interactions reveal both a canonical and an atypical site to be occupied by the anionic lipid. PH domain binding leads to local clustering of PIP molecules in the bilayer. Association of the Dok7 PH domain with PIP lipids is therefore seen as a key step in localization of Dok7 to the membrane and formation of a complex with MuSK.

The B-domain of factor VIII reduces cell membrane attachement to host cells in serum free conditions

DEFF Research Database (Denmark)

Kolind, Mille Petersen; Nørby, Peder Lisby; Flintegaard, Thomas Veje

2010-01-01

engineered extensively throughout the years to increase the low production yields that initially were obtained from mammalian cell cultures. The scope of this work was to investigate the interaction of rFVIII with the cell membrane surface of the producing cells in serum free medium. We wondered whether...... binding of rFVIII to the cell membrane could be a factor diminishing the production yield. We studied the contribution of the rFVIII B-domain to membrane attachment by transfecting several constructs containing increasing lengths of the B-domain into cells under serum free conditions. We found that 90......% of rFVIII is attached to the cell membrane of the producing cell when the rFVIII variant contains a short B-domain (21 aa). By increasing the length of the B-domain the membrane attached fraction can be reduced to 50% of the total expressed rFVIII. Further, our studies show that the N...

Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains

DEFF Research Database (Denmark)

Bækgaard, Lone; Fuglsang, Anja Thoe; Palmgren, Michael Gjedde

2005-01-01

In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H(+)-and Ca(2+)-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory...... mechanisms of these pumps. In plant plasma membrane H(+)- and Ca(2+)-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed....

Holographic QCD with topologically charged domain-wall/membranes

International Nuclear Information System (INIS)

Lin Fengli; Wu Shangyu

2008-01-01

We study the thermodynamical phase structures of holographic QCD with nontrivial topologically charged domain-wall/membranes which are originally related to the multiple θ-vacua in the large N c limit. We realize the topologically charged membranes as the holographic D6-brane fluxes in the Sakai-Sugimoto model. The D6-brane fluxes couple to the probe D8-D8-bar via Chern-Simon term, and act as the source for the baryonic current density of QCD. We find rich phase structures of the dual meson system by varying asymptotic separation of D8 and D8-bar. Especially, there can be a thermodynamically favored and stable phase of finite baryonic current density. This provides the supporting evidence for the discovery of the topologically charged membranes found in the lattice QCD calculations. We also find a crossover phase with the limiting baryonic current density and temperature which suggest a Hagedorn-like phase transition of meson dissociation.

Monitoring Protein Fouling on Polymeric Membranes Using Ultrasonic Frequency-Domain Reflectometry

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Robin Fong

2011-08-01

Full Text Available Novel signal-processing protocols were used to extend the in situ sensitivity of ultrasonic frequency-domain reflectometry (UFDR for real-time monitoring of microfiltration (MF membrane fouling during protein purification. Different commercial membrane materials, with a nominal pore size of 0.2 µm, were challenged using bovine serum albumin (BSA and amylase as model proteins. Fouling induced by these proteins was observed in flat-sheet membrane filtration cells operating in a laminar cross-flow regime. The detection of membrane-associated proteins using UFDR was determined by applying rigorous statistical methodology to reflection spectra of ultrasonic signals obtained during membrane fouling. Data suggest that the total power reflected from membrane surfaces changes in response to protein fouling at concentrations as low as 14 μg/cm2, and results indicate that ultrasonic spectra can be leveraged to detect and monitor protein fouling on commercial MF membranes.

Super-resolution optical microscopy for studying membrane structure and dynamics.

Science.gov (United States)

Sezgin, Erdinc

2017-07-12

Investigation of cell membrane structure and dynamics requires high spatial and temporal resolution. The spatial resolution of conventional light microscopy is limited due to the diffraction of light. However, recent developments in microscopy enabled us to access the nano-scale regime spatially, thus to elucidate the nanoscopic structures in the cellular membranes. In this review, we will explain the resolution limit, address the working principles of the most commonly used super-resolution microscopy techniques and summarise their recent applications in the biomembrane field.

Compartmentalized cAMP Signaling Associated With Lipid Raft and Non-raft Membrane Domains in Adult Ventricular Myocytes.

Science.gov (United States)

Agarwal, Shailesh R; Gratwohl, Jackson; Cozad, Mia; Yang, Pei-Chi; Clancy, Colleen E; Harvey, Robert D

2018-01-01

Aim: Confining cAMP production to discrete subcellular locations makes it possible for this ubiquitous second messenger to elicit unique functional responses. Yet, factors that determine how and where the production of this diffusible signaling molecule occurs are incompletely understood. The fluid mosaic model originally proposed that signal transduction occurs through random interactions between proteins diffusing freely throughout the plasma membrane. However, it is now known that the movement of membrane proteins is restricted, suggesting that the plasma membrane is segregated into distinct microdomains where different signaling proteins can be concentrated. In this study, we examined what role lipid raft and non-raft membrane domains play in compartmentation of cAMP signaling in adult ventricular myocytes. Methods and Results: The freely diffusible fluorescence resonance energy transfer-based biosensor Epac2-camps was used to measure global cytosolic cAMP responses, while versions of the probe targeted to lipid raft (Epac2-MyrPalm) and non-raft (Epac2-CAAX) domains were used to monitor local cAMP production near the plasma membrane. We found that β-adrenergic receptors, which are expressed in lipid raft and non-raft domains, produce cAMP responses near the plasma membrane that are distinctly different from those produced by E-type prostaglandin receptors, which are expressed exclusively in non-raft domains. We also found that there are differences in basal cAMP levels associated with lipid raft and non-raft domains, and that this can be explained by differences in basal adenylyl cyclase activity associated with each of these membrane environments. In addition, we found evidence that phosphodiesterases 2, 3, and 4 work together in regulating cAMP activity associated with both lipid raft and non-raft domains, while phosphodiesterase 3 plays a more prominent role in the bulk cytoplasmic compartment. Conclusion: These results suggest that different membrane

Activation of Endothelial Nitric Oxide (eNOS Occurs through Different Membrane Domains in Endothelial Cells.

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Jason Tran

Full Text Available Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC with cholesterol and the oxysterol 7-ketocholesterol (7KC. Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1 colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.

Nanoscale protein architecture of the kidney glomerular basement membrane

Science.gov (United States)

Suleiman, Hani; Zhang, Lei; Roth, Robyn; Heuser, John E; Miner, Jeffrey H; Shaw, Andrey S; Dani, Adish

2013-01-01

In multicellular organisms, proteins of the extracellular matrix (ECM) play structural and functional roles in essentially all organs, so understanding ECM protein organization in health and disease remains an important goal. Here, we used sub-diffraction resolution stochastic optical reconstruction microscopy (STORM) to resolve the in situ molecular organization of proteins within the kidney glomerular basement membrane (GBM), an essential mediator of glomerular ultrafiltration. Using multichannel STORM and STORM-electron microscopy correlation, we constructed a molecular reference frame that revealed a laminar organization of ECM proteins within the GBM. Separate analyses of domains near the N- and C-termini of agrin, laminin, and collagen IV in mouse and human GBM revealed a highly oriented macromolecular organization. Our analysis also revealed disruptions in this GBM architecture in a mouse model of Alport syndrome. These results provide the first nanoscopic glimpse into the organization of a complex ECM. DOI: http://dx.doi.org/10.7554/eLife.01149.001 PMID:24137544

Giant Plasma Membrane Vesicles: An Experimental Tool for Probing the Effects of Drugs and Other Conditions on Membrane Domain Stability.

Science.gov (United States)

Gerstle, Zoe; Desai, Rohan; Veatch, Sarah L

2018-01-01

Giant plasma membrane vesicles (GPMVs) are isolated directly from living cells and provide an alternative to vesicles constructed of synthetic or purified lipids as an experimental model system for use in a wide range of assays. GPMVs capture much of the compositional protein and lipid complexity of intact cell plasma membranes, are filled with cytoplasm, and are free from contamination with membranes from internal organelles. GPMVs often exhibit a miscibility transition below the growth temperature of their parent cells. GPMVs labeled with a fluorescent protein or lipid analog appear uniform on the micron-scale when imaged above the miscibility transition temperature, and separate into coexisting liquid domains with differing membrane compositions and physical properties below this temperature. The presence of this miscibility transition in isolated GPMVs suggests that a similar phase-like heterogeneity occurs in intact plasma membranes under growth conditions, albeit on smaller length scales. In this context, GPMVs provide a simple and controlled experimental system to explore how drugs and other environmental conditions alter the composition and stability of phase-like domains in intact cell membranes. This chapter describes methods to generate and isolate GPMVs from adherent mammalian cells and to interrogate their miscibility transition temperatures using fluorescence microscopy. © 2018 Elsevier Inc. All rights reserved.

Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain.

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Danthi, Pranav; Coffey, Caroline M; Parker, John S L; Abel, Ty W; Dermody, Terence S

2008-12-01

Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein mu1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of mu1 indicates that the mu1 phi domain is sufficient to elicit a cell death response. To evaluate the contribution of the mu1 phi domain to the induction of apoptosis following reovirus infection, phi mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective phi mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain.

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Pranav Danthi

2008-12-01

Full Text Available Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein mu1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of mu1 indicates that the mu1 phi domain is sufficient to elicit a cell death response. To evaluate the contribution of the mu1 phi domain to the induction of apoptosis following reovirus infection, phi mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective phi mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

Science.gov (United States)

Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

2014-01-21

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Phospho-Caveolin-1 Mediates Integrin-Regulated Membrane Domain Internalisation

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del Pozo, Miguel A.; Alderson, Nazilla B.; Grande-García, Araceli; Balasubramanian, Nagaraj; Schwartz, Martin A.; Kiosses, William B.; Anderson, Richard G.W.

2005-01-01

Growth of normal cells is anchorage-dependent because signalling through multiple pathways including Erk, PI 3-kinase and Rac requires integrin-mediated cell adhesion 1. Components of these pathways localize to low density, cholesterol-rich domains in the plasma membrane named “lipid rafts” 2,3 or “cholesterol enriched membrane microdomains” (CEMM) 4. We previously reported that integrin-mediated adhesion regulates CEMM trafficking such that cell detachment from the extracellular matrix (ECM) triggers CEMM internalisation and clearance from the plasma membrane 5. We now report that this internalisation is mediated by dynamin-2 and caveolin-1. Internalisation requires phosphorylation of caveolin-1 on tyrosine 14. A shift in localisation of phospho-caveolin-1 from focal adhesions to caveolae induces CEMM internalisation upon cell detachment, which mediates inhibition of Erk, PI 3-kinase and Rac. These data define a novel molecular mechanism for growth and tumour suppression by caveolin-1. PMID:16113676

An Amphipathic Helix Directs Cellular Membrane Curvature Sensing and Function of the BAR Domain Protein PICK1.

Science.gov (United States)

Herlo, Rasmus; Lund, Viktor K; Lycas, Matthew D; Jansen, Anna M; Khelashvili, George; Andersen, Rita C; Bhatia, Vikram; Pedersen, Thomas S; Albornoz, Pedro B C; Johner, Niklaus; Ammendrup-Johnsen, Ina; Christensen, Nikolaj R; Erlendsson, Simon; Stoklund, Mikkel; Larsen, Jannik B; Weinstein, Harel; Kjærulff, Ole; Stamou, Dimitrios; Gether, Ulrik; Madsen, Kenneth L

2018-05-15

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Lipid domains in intact fiber-cell plasma membranes isolated from cortical and nuclear regions of human eye lenses of donors from different age groups.

Science.gov (United States)

Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

2015-03-01

The results reported here clearly document changes in the properties and the organization of fiber-cell membrane lipids that occur with age, based on electron paramagnetic resonance (EPR) analysis of lens membranes of clear lenses from donors of age groups from 0 to 20, 21 to 40, and 61 to 80 years. The physical properties, including profiles of the alkyl chain order, fluidity, hydrophobicity, and oxygen transport parameter, were investigated using EPR spin-labeling methods, which also provide an opportunity to discriminate coexisting lipid domains and to evaluate the relative amounts of lipids in these domains. Fiber-cell membranes were found to contain three distinct lipid environments: bulk lipid domain, which appears minimally affected by membrane proteins, and two domains that appear due to the presence of membrane proteins, namely boundary and trapped lipid domains. In nuclear membranes the amount of boundary and trapped phospholipids as well as the amount of cholesterol in trapped lipid domains increased with the donors' age and was greater than that in cortical membranes. The difference between the amounts of lipids in domains uniquely formed due to the presence of membrane proteins in nuclear and cortical membranes increased with the donors' age. It was also shown that cholesterol was to a large degree excluded from trapped lipid domains in cortical membranes. It is evident that the rigidity of nuclear membranes was greater than that of cortical membranes for all age groups. The amount of lipids in domains of low oxygen permeability, mainly in trapped lipid domains, were greater in nuclear than cortical membranes and increased with the age of donors. These results indicate that the nuclear fiber cell plasma membranes were less permeable to oxygen than cortical membranes and become less permeable to oxygen with age. In clear lenses, age-related changes in the lens lipid and protein composition and organization appear to occur in ways that increase fiber

Salt Bridge Formation between the I-BAR Domain and Lipids Increases Lipid Density and Membrane Curvature.

Science.gov (United States)

Takemura, Kazuhiro; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro; Kitao, Akio

2017-07-28

The BAR domain superfamily proteins sense or induce curvature in membranes. The inverse-BAR domain (I-BAR) is a BAR domain that forms a straight "zeppelin-shaped" dimer. The mechanisms by which IRSp53 I-BAR binds to and deforms a lipid membrane are investigated here by all-atom molecular dynamics simulation (MD), binding energy analysis, and the effects of mutation experiments on filopodia on HeLa cells. I-BAR adopts a curved structure when crystallized, but adopts a flatter shape in MD. The binding of I-BAR to membrane was stabilized by ~30 salt bridges, consistent with experiments showing that point mutations of the interface residues have little effect on the binding affinity whereas multiple mutations have considerable effect. Salt bridge formation increases the local density of lipids and deforms the membrane into a concave shape. In addition, the point mutations that break key intra-molecular salt bridges within I-BAR reduce the binding affinity; this was confirmed by expressing these mutants in HeLa cells and observing their effects. The results indicate that the stiffness of I-BAR is important for membrane deformation, although I-BAR does not act as a completely rigid template.

Domains of increased thickness in microvillar membranes of the small intestinal enterocyte

DEFF Research Database (Denmark)

Kunding, Andreas H; Christensen, Sune M; Danielsen, E Michael

2010-01-01

The apical surface of the enterocyte is sculpted into a dense array of cylindrical microvillar protrusions by supporting actin filaments. Membrane microdomains (rafts) enriched in cholesterol and glycosphingolipids comprise roughly 50% of the microvillar membrane and play a vital role in orchestr......The apical surface of the enterocyte is sculpted into a dense array of cylindrical microvillar protrusions by supporting actin filaments. Membrane microdomains (rafts) enriched in cholesterol and glycosphingolipids comprise roughly 50% of the microvillar membrane and play a vital role...... in orchestrating absorptive/digestive action of dietary nutrients at this important cellular interface. Increased membrane thickness is believed to be a morphological characteristic of rafts. Thus, we investigated whether the high contents of lipid rafts in the microvillar membrane is reflected in local variations...... was clearly monophasic. The encountered domains of increased thickness (DITs) occupied 48% of the microvillar membrane and from the data we estimated the area of a single DIT to have a lower limit of 600 nm(2). In other experiments we mapped the organization of biochemically defined lipid rafts by immunogold...

Plasma membrane partitioning: from macro-domains to new views on plasmodesmata

Directory of Open Access Journals (Sweden)

Yohann eBoutté

2014-04-01

Full Text Available Compartmentalization of cellular functions relies on partitioning of domains of diverse sizes within the plasma membrane (PM. Macro-domains measure several micrometers and contain specific proteins concentrated to specific sides (apical, basal and lateral of the PM conferring a polarity to the cell. Cell polarity is one of the driving forces in tissue and growth patterning. To maintain macro-domains within the PM, eukaryotic cells exert diverse mechanisms to counteract the free lateral diffusion of proteins. Protein activation/inactivation, endocytosis, PM recycling of transmembrane proteins and the role of diffusion barriers in macro-domains partitioning at PM will be discussed. Moreover, as plasmodesmata (PDs are domains inserted within the PM which also mediate tissue and growth patterning, it is essential to understand how segregation of specific set of proteins is maintained at PDs while PDs domains are smaller in size compared to macro-domains. Here, we will present mechanisms allowing restriction of proteins at PM macrodomains, but for which molecular components have been found in PDs proteome. We will explore the hypothesis that partitioning of macro-domains and PDs may be ruled by similar mechanisms.

Membrane Restructuring by Phospholipase A2 Is Regulated by the Presence of Lipid Domains

DEFF Research Database (Denmark)

Leidy, Chad; Ocampo, Jackson; Duelund, Lars

2011-01-01

Secretory phospholipase A2 (sPLA2) catalyzes the hydrolysis of glycerophospholipids. This enzyme is sensitive to membrane structure, and its activity has been shown to increase in the presence of liquid-crystalline/gel (Lα/Lβ) lipid domains. In this work, we explore whether lipid domains can also...... without necessarily destroying the membrane. We confirm by high-performance liquid chromatography the preferential hydrolysis of DMPC within the phase coexistence region of the DMPC/DSPC phase diagram, showing that this preferential hydrolysis is accentuated close to the solidus phase boundary...

Spontaneous insertion of GPI anchors into cholesterol-rich membrane domains

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Jing Li

2018-05-01

Full Text Available GPI-Anchored proteins (GPI-APs can be exogenously transferred onto bilayer membranes both in vivo and in vitro, while the mechanism by which this transfer process occurs is unknown. In this work, we used atomistic molecular dynamics simulations and free energy calculations to characterize the essential influence of cholesterol on insertion of the GPI anchors into plasma membranes. We demonstrate, both dynamically and energetically, that in the presence of cholesterol, the tails of GPI anchors are able to penetrate inside the core of the lipid membrane spontaneously with a three-step mechanism, while in the absence of cholesterol no spontaneous insertion was observed. We ascribe the failure of insertion to the strong thermal fluctuation of lipid molecules in cholesterol-free bilayer, which generates a repulsive force in entropic origin. In the presence of cholesterol, however, the fluctuation of lipids is strongly reduced, thus decreasing the barrier for the anchor insertion. Based on this observation, we propose a hypothesis that addition of cholesterol creates vertical creases in membranes for the insertion of acyl chains. Moreover, we find that the GPI anchor could also spontaneously inserted into the boundary between cholesterol-rich and cholesterol-depleted domains. Our results shed light on the mechanism of cholesterol-mediated interaction between membrane proteins with acyl chain and plasma membranes in living cells.

Spontaneous insertion of GPI anchors into cholesterol-rich membrane domains

Science.gov (United States)

Li, Jing; Liu, Xiuhua; Tian, Falin; Yue, Tongtao; Zhang, Xianren; Cao, Dapeng

2018-05-01

GPI-Anchored proteins (GPI-APs) can be exogenously transferred onto bilayer membranes both in vivo and in vitro, while the mechanism by which this transfer process occurs is unknown. In this work, we used atomistic molecular dynamics simulations and free energy calculations to characterize the essential influence of cholesterol on insertion of the GPI anchors into plasma membranes. We demonstrate, both dynamically and energetically, that in the presence of cholesterol, the tails of GPI anchors are able to penetrate inside the core of the lipid membrane spontaneously with a three-step mechanism, while in the absence of cholesterol no spontaneous insertion was observed. We ascribe the failure of insertion to the strong thermal fluctuation of lipid molecules in cholesterol-free bilayer, which generates a repulsive force in entropic origin. In the presence of cholesterol, however, the fluctuation of lipids is strongly reduced, thus decreasing the barrier for the anchor insertion. Based on this observation, we propose a hypothesis that addition of cholesterol creates vertical creases in membranes for the insertion of acyl chains. Moreover, we find that the GPI anchor could also spontaneously inserted into the boundary between cholesterol-rich and cholesterol-depleted domains. Our results shed light on the mechanism of cholesterol-mediated interaction between membrane proteins with acyl chain and plasma membranes in living cells.

The C-terminal domain of TRPV4 is essential for plasma membrane localization.

Science.gov (United States)

Becker, Daniel; Müller, Margarethe; Leuner, Kristina; Jendrach, Marina

2008-02-01

Many members of the TRP superfamily oligomerize in the ER before trafficking to the plasma membrane. For membrane localization of the non-selective cation channel TRPV4 specific domains in the N-terminus are required, but the role of the C-terminus in the oligomerization and trafficking process has been not determined until now. Therefore, the localization of recombinant TRPV4 in two cell models was analyzed: HaCaT keratinocytes that express TRPV4 endogenously were compared to CHO cells that are devoid of endogenous TRPV4. When deletions were introduced in the C-terminal domain three states of TRPV4 localization were defined: a truncated TRPV4 protein of 855 amino acids was exported to the plasma membrane like the full-length channel (871 aa) and was also functional. Mutants with a length of 828 to 844 amino acids remained in the ER of CHO cells, but in HaCaT cells plasma membrane localization was partially rescued by oligomerization with endogenous TRPV4. This was confirmed by coexpression of recombinant full-length TRPV4 together with these deletion mutants, which resulted in an almost complete plasma membrane localization of both proteins and significant FRET in the plasma membrane and the ER. All deletions upstream of amino acid 828 resulted in total ER retention that could not rescued by coexpression with the full-length protein. However, these deletion mutants did not impair export of full-length TRPV4, implying that no oligomerization took place. These data indicate that the C-terminus of TRPV4 is required for oligomerization, which takes place in the ER and precedes plasma membrane trafficking.

Diffusion of flexible, charged, nanoscopic molecules in solution: Size and pH dependence for PAMAM dendrimer

Science.gov (United States)

Maiti, Prabal K.; Bagchi, Biman

2009-12-01

In order to understand self-diffusion (D) of a charged, flexible, and porous nanoscopic molecule in water, we carry out very long, fully atomistic molecular dynamics simulation of PAMAM dendrimer up to eight generations in explicit salt water under varying pH. We find that while the radius of gyration (Rg) varies as N1/3, the self-diffusion constant (D ) scales, surprisingly, as N-α, with α =0.39 at high pH and 0.5 at neutral pH, indicating a dramatic breakdown of Stokes-Einstein relation for diffusion of charged nanoscopic molecules. The variation in D as a function of radius of gyration demonstrates the importance of treating water and ions explicitly in the diffusion process of a flexible nanoscopic molecule. In agreement with recent experiments, the self-diffusion constant increases with pH, revealing the importance of dielectric friction in the diffusion process. The shape of a dendrimer is found to fluctuate on a nanosecond time scale. We argue that this flexibility (and also the porosity) of the dendrimer may play an important role in determining the mean square displacement of the dendrimer and the breakdown of the Stokes-Einstein relation between diffusion constant and the radius.

With or without rafts? Alternative views on cell membranes.

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Sevcsik, Eva; Schütz, Gerhard J

2016-02-01

The fundamental mechanisms of protein and lipid organization at the plasma membrane have continued to engage researchers for decades. Among proposed models, one idea has been particularly successful which assumes that sterol-dependent nanoscopic phases of different lipid chain order compartmentalize proteins, thereby modulating protein functionality. This model of membrane rafts has sustainably sparked the fields of membrane biophysics and biology, and shifted membrane lipids into the spotlight of research; by now, rafts have become an integral part of our terminology to describe a variety of cell biological processes. But is the evidence clear enough to continue supporting a theoretical concept which has resisted direct proof by observation for nearly twenty years? In this essay, we revisit findings that gave rise to and substantiated the raft hypothesis, discuss its impact on recent studies, and present alternative mechanisms to account for plasma membrane heterogeneity. © 2015 WILEY Periodicals, Inc.

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains*

Science.gov (United States)

Yoon, Youngdae; Zhang, Xiuqi; Cho, Wonhwa

2012-01-01

Cellular proteins containing Bin/amphiphysin/Rvs (BAR) domains play a key role in clathrin-mediated endocytosis. Despite extensive structural and functional studies of BAR domains, it is still unknown how exactly these domains interact with the plasma membrane containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P2 specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H0) of two representative N-BAR domains from Drosophila amphiphysin (dAmp-BAR) and rat endophilin A1 (EndoA1-BAR). Our quantitative fluorescence imaging analysis shows that PtdIns(4,5)P2-dependent membrane penetration of H0 is important for self-association of membrane-bound dAmp-BAR and EndoA1-BAR and their membrane deformation activity. EndoA1-BAR behaves differently from dAmp-BAR because the former has an additional amphiphilic α-helix that penetrates the membrane in a PtdIns(4,5)P2-independent manner. Depletion of PtdIns(4,5)P2 from the plasma membrane of HEK293 cells abrogated the membrane deforming activity of EndoA1-BAR and dAmp-BAR. Collectively, these studies suggest that the local PtdIns(4,5)P2 concentration in the plasma membrane may regulate the membrane interaction and deformation by N-BAR domain-containing proteins during clathrin-mediated endocytosis. PMID:22888025

Co-existence of Gel and Fluid Lipid Domains in Single-component Phospholipid Membranes

Energy Technology Data Exchange (ETDEWEB)

Armstrong, Clare L [McMaster University; Barrett, M [McMaster University; Toppozini, L [McMaster University; Yamani, Zahra [Canadian Neutron Beam Centre, National Research Council, Chalk River Laboratorie; Kucerka, Norbert [Canadian Neutron Beam Centre and Comelius University (Slovakia); Katsaras, John [ORNL; Fragneto, Giovanna [Institut Laue-Langevin (ILL); Rheinstadter, Maikel C [McMaster University

2012-01-01

Lateral nanostructures in membranes, so-called rafts, are believed to strongly influence membrane properties and functions. The experimental observation of rafts has proven difficult as they are thought to be dynamic structures that likely fluctuate on nano- to microsecond time scales. Using neutron diffraction we present direct experimental evidence for the co-existence of gel and fluid lipid domains in a single-component phospholipid membrane made of DPPC as it undergoes its main phase transition. The coherence length of the neutron beam sets a lower limit for the size of structures that can be observed. Neutron coherence lengths between 30 and 242A used in this study were obtained by varying the incident neutron energy and the resolution of the neutron spectrometer. We observe Bragg peaks corresponding to co-existing nanometer sized structures, both in out-of-plane and in-plane scans, by tuning the neutron coherence length. During the main phase transition, instead of a continuous transition that shows a pseudo-critical behavior, we observe the co-existence of gel and fluid domains.

Insight into the adsorption profiles of the Saprolegnia monoica chitin synthase MIT domain on POPA and POPC membranes by molecular dynamics simulation studies.

Science.gov (United States)

Kuang, Guanglin; Liang, Lijun; Brown, Christian; Wang, Qi; Bulone, Vincent; Tu, Yaoquan

2016-02-21

The critical role of chitin synthases in oomycete hyphal tip growth has been established. A microtubule interacting and trafficking (MIT) domain was discovered in the chitin synthases of the oomycete model organism, Saprolegnia monoica. MIT domains have been identified in diverse proteins and may play a role in intracellular trafficking. The structure of the Saprolegnia monoica chitin synthase 1 (SmChs1) MIT domain has been recently determined by our group. However, although our in vitro assay identified increased strength in interactions between the MIT domain and phosphatidic acid (PA) relative to other phospholipids including phosphatidylcholine (PC), the mechanism used by the MIT domain remains unknown. In this work, the adsorption behavior of the SmChs1 MIT domain on POPA and POPC membranes was systematically investigated by molecular dynamics simulations. Our results indicate that the MIT domain can adsorb onto the tested membranes in varying orientations. Interestingly, due to the specific interactions between MIT residues and lipid molecules, the binding affinity to the POPA membrane is much higher than that to the POPC membrane. A binding hotspot, which is critical for the adsorption of the MIT domain onto the POPA membrane, was also identified. The lower binding affinity to the POPC membrane can be attributed to the self-saturated membrane surface, which is unfavorable for hydrogen-bond and electrostatic interactions. The present study provides insight into the adsorption profile of SmChs1 and additionally has the potential to improve our understanding of other proteins containing MIT domains.

A novel form of the membrane protein CD147 that contains an extra Ig-like domain and interacts homophilically

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Brown Marion H

2003-11-01

Full Text Available Abstract Background CD147 is a broadly distributed integral membrane glycoprotein with two Ig-like domains implicated in a wide range of functions. It is associated at the cell surface with the monocarboxylate transporters MCT1 and 4 but interactions of the extracellular region have not been characterised. Results We report the characterisation of a form of CD147 with an additional membrane-distal Ig-like domain. In contrast to the two domain form, this three domain form of CD147 interacts homophilically. Surface plasmon resonance analysis using recombinant proteins showed that the interaction was of low affinity (KD ~ 40 μM and this is typical of many interactions between membrane proteins. cDNA for the 3 domain form are rare but have been identified in human and mouse retina. Conclusion The finding that the three domain form of CD147 has an extracellular ligand, that is it interacts homophilically, suggests this interaction may be important in aligning lactate transporters in the retina where lactate is an important metabolite.

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

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Zárský, Viktor; Potocký, Martin

2010-04-01

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

The F-BAR domains from srGAP1, srGAP2 and srGAP3 regulate membrane deformation differently

Science.gov (United States)

Coutinho-Budd, Jaeda; Ghukasyan, Vladimir; Zylka, Mark J.; Polleux, Franck

2012-01-01

Summary Coordination of membrane deformation and cytoskeletal dynamics lies at the heart of many biological processes critical for cell polarity, motility and morphogenesis. We have recently shown that Slit-Robo GTPase-activating protein 2 (srGAP2) regulates neuronal morphogenesis through the ability of its F-BAR domain to regulate membrane deformation and induce filopodia formation. Here, we demonstrate that the F-BAR domains of two closely related family members, srGAP1 and srGAP3 [designated F-BAR(1) and F-BAR(3), respectively] display significantly different membrane deformation properties in non-neuronal COS7 cells and in cortical neurons. F-BAR(3) induces filopodia in both cell types, though less potently than F-BAR(2), whereas F-BAR(1) prevents filopodia formation in cortical neurons and reduces plasma membrane dynamics. These three F-BAR domains can heterodimerize, and they act synergistically towards filopodia induction in COS7 cells. As measured by fluorescence recovery after photobleaching, F-BAR(2) displays faster molecular dynamics than F-BAR(3) and F-BAR(1) at the plasma membrane, which correlates well with its increased potency to induce filopodia. We also show that the molecular dynamic properties of F-BAR(2) at the membrane are partially dependent on F-Actin. Interestingly, acute phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] depletion in cells does not interfere with plasma membrane localization of F-BAR(2), which is compatible with our result showing that F-BAR(2) binds to a broad range of negatively-charged phospholipids present at the plasma membrane, including phosphatidylserine (PtdSer). Overall, our results provide novel insights into the functional diversity of the membrane deformation properties of this subclass of F-BAR-domains required for cell morphogenesis. PMID:22467852

GRP1 PH Domain, Like AKT1 PH Domain, Possesses a Sentry Glutamate Residue Essential for Specific Targeting to Plasma Membrane PI(3,4,5)P3

Science.gov (United States)

Pilling, Carissa; Landgraf, Kyle E.; Falke, Joseph J.

2011-01-01

During the appearance of the signaling lipid PI(3,4,5)P3, an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P3-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P2 and bind the rare PI(3,4,5)P3 target lipid with sufficiently high affinity. Our previous study of the E17K mutant of protein kinase B (AKT1) PH domain, together with evidence from Carpten et al (1), revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P2, thereby playing an essential role in specific PI(3,4,5)P3 targeting (2). The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P3-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P2 affinity and constitutive plasma membrane targeting. To test this hypothesis the present study investigates the E345 residue, a putative sentry glutamate, of General Receptor for Phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into GRP1 PH domain enhances PI(4,5)P2 affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P2 releases E345K GRP1 PH domain into the cytoplasm and the efficiency of this release increases when target Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K (1, 3). Analysis of available PH domain structures suggests that a lone glutamate residue (or, in some cases an aspartate) is a common, perhaps ubiquitous, feature of PI(3,4,5)P3-specific binding

Diffusion mediated coagulation and fragmentation based study of domain formation in lipid bilayer membrane

Energy Technology Data Exchange (ETDEWEB)

Rao, Laxminarsimha V., E-mail: laxman@iitk.ac.in [Mechanics and Applied Mathematics Group, Department of Mechanical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India); Roy, Subhradeep [Department of Biomedical Engineering and Mechanics (MC 0219), Virginia Tech, 495 Old Turner Street, Blacksburg, VA 24061 (United States); Das, Sovan Lal [Mechanics and Applied Mathematics Group, Department of Mechanical Engineering, Indian Institute of Technology Kanpur, Kanpur 208016 (India)

2017-01-15

We estimate the equilibrium size distribution of cholesterol rich micro-domains on a lipid bilayer by solving Smoluchowski equation for coagulation and fragmentation. Towards this aim, we first derive the coagulation kernels based on the diffusion behaviour of domains moving in a two dimensional membrane sheet, as this represents the reality better. We incorporate three different diffusion scenarios of domain diffusion into our coagulation kernel. Subsequently, we investigate the influence of the parameters in our model on the coagulation and fragmentation behaviour. The observed behaviours of the coagulation and fragmentation kernels are also manifested in the equilibrium domain size distribution and its first moment. Finally, considering the liquid domains diffusing in a supported lipid bilayer, we fit the equilibrium domain size distribution to a benchmark solution.

Physics of zero- and one-dimensional nanoscopic systems

CERN Document Server

Maiti, Santanu; Chowdhury, Jayeeta

2007-01-01

In recent years submicron and nanoscale systems have featured strongly on the research agenda due to the technological progress and new physics that have emerged from studies of ultra-small systems. A fundamental understanding of basic physical phenomena on the mesoscopic and nanoscopic scales is required to exploit the technological potential offered by these exotic materials. The present book contains review-like chapters by some of the leading experts in the field, covering topics such as the Kondo effect, electron transport, disorder and quantum coherence with electron-electron interaction, persistent current, thermoelectric phenomena, etc. in quantum dots, quantum wires, carbon nanotubes and more. This book will be valuable to researchers and students in condensed matter physics.

Promiscuous and specific phospholipid binding by domains in ZAC, a membrane-associated Arabidopsis protein with an ARF GAP zinc finger and a C2 domain

DEFF Research Database (Denmark)

Jensen, R B; Lykke-Andersen, K; Frandsen, G I

2000-01-01

domain are separated by a region without homology to other known proteins. Zac promoter/beta-glucuronidase reporter assays revealed highest expression levels in flowering tissue, rosettes and roots. ZAC protein was immuno-detected mainly in association with membranes and fractionated with Golgi...... and plasma membrane marker proteins. ZAC membrane association was confirmed in assays by a fusion between ZAC and the green fluorescence protein and prompted an analysis of the in vitro phospholipid-binding ability of ZAC. Phospholipid dot-blot and liposome-binding assays indicated that fusion proteins...... zinc finger motif, but proteins containing only the zinc finger domain (residues 1-105) did not bind PI-3-P. Recombinant ZAC possessed GTPase-activating activity on Arabidopsis ARF proteins. These data identify a novel PI-3-P-binding protein region and thereby provide evidence...

Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP)

International Nuclear Information System (INIS)

Terawaki, Shin-ichi; Kitano, Ken; Aoyama, Miki; Hakoshima, Toshio

2008-01-01

The radixin FERM domain was shown to bind the MT1-MMP cytoplasmic peptide and crystals of the complex were obtained. ERM proteins play a role in the cross-linking found between plasma membranes and actin filaments. The N-terminal FERM domains of ERM proteins are responsible for membrane association through direct interaction with the cytoplasmic tails of integral membrane proteins. During cell migration and movement, membrane-type 1 matrix metalloproteinase (MT1-MMP) on plasma membranes sheds adhesion molecule CD44 in addition to degrading the extracellular matrix. Here, the interaction between the radixin FERM domain and the MT1-MMP cytoplasmic tail is reported and preliminary crystallographic characterization of crystals of the radixin FERM domain bound to the cytoplasmic tail of MT1-MMP is presented. The crystals belong to space group P6 1 22, with unit-cell parameters a = b = 122.7, c = 128.3 Å, and contain one complex in the crystallographic asymmetric unit. The diffraction data were collected to a resolution of 2.4 Å

A Novel Plasma Membrane-Anchored Protein Regulates Xylem Cell-Wall Deposition through Microtubule-Dependent Lateral Inhibition of Rho GTPase Domains.

Science.gov (United States)

Sugiyama, Yuki; Wakazaki, Mayumi; Toyooka, Kiminori; Fukuda, Hiroo; Oda, Yoshihisa

2017-08-21

Spatial control of cell-wall deposition is essential for determining plant cell shape [1]. Rho-type GTPases, together with the cortical cytoskeleton, play central roles in regulating cell-wall patterning [2]. In metaxylem vessel cells, which are the major components of xylem tissues, active ROP11 Rho GTPases form oval plasma membrane domains that locally disrupt cortical microtubules, thereby directing the formation of oval pits in secondary cell walls [3-5]. However, the regulatory mechanism that determines the planar shape of active Rho of Plants (ROP) domains is still unknown. Here we show that IQD13 associates with cortical microtubules and the plasma membrane to laterally restrict the localization of ROP GTPase domains, thereby directing the formation of oval secondary cell-wall pits. Loss and overexpression of IQD13 led to the formation of abnormally round and narrow secondary cell-wall pits, respectively. Ectopically expressed IQD13 increased the presence of parallel cortical microtubules by promoting microtubule rescue. A reconstructive approach revealed that IQD13 confines the area of active ROP domains within the lattice of the cortical microtubules, causing narrow ROP domains to form. This activity required the interaction of IQD13 with the plasma membrane. These findings suggest that IQD13 positively regulates microtubule dynamics as well as their linkage to the plasma membrane, which synergistically confines the area of active ROP domains, leading to the formation of oval secondary cell-wall pits. This finding sheds light on the role of microtubule-plasma membrane linkage as a lateral fence that determines the planar shape of Rho GTPase domains. Copyright © 2017 Elsevier Ltd. All rights reserved.

Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study.

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Huai-Chun Chen

Full Text Available The second messenger lipid PIP(3 (phosphatidylinositol-3,4,5-trisphosphate is generated by the lipid kinase PI3K (phosphoinositide-3-kinase in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3-specific pleckstrin homology (PH domains to the membrane surface. Despite the broad importance of PIP(3-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3 lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3. The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i PIP(3 target lipid that provides specificity and affinity, and (ii PS facilitator lipid that enhances the PIP(3 on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3 headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3 headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3 headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral

The topogenic function of S4 promotes membrane insertion of the voltage-sensor domain in the KvAP channel.

Science.gov (United States)

Mishima, Eriko; Sato, Yoko; Nanatani, Kei; Hoshi, Naomi; Lee, Jong-Kook; Schiller, Nina; von Heijne, Gunnar; Sakaguchi, Masao; Uozumi, Nobuyuki

2016-12-01

Voltage-dependent K + (K V ) channels control K + permeability in response to shifts in the membrane potential. Voltage sensing in K V channels is mediated by the positively charged transmembrane domain S4. The best-characterized K V channel, KvAP, lacks the distinct hydrophilic region corresponding to the S3-S4 extracellular loop that is found in other K + channels. In the present study, we evaluated the topogenic properties of the transmembrane regions within the voltage-sensing domain in KvAP. S3 had low membrane insertion activity, whereas S4 possessed a unique type-I signal anchor (SA-I) function, which enabled it to insert into the membrane by itself. S4 was also found to function as a stop-transfer signal for retention in the membrane. The length and structural nature of the extracellular S3-S4 loop affected the membrane insertion of S3 and S4, suggesting that S3 membrane insertion was dependent on S4. Replacement of charged residues within the transmembrane regions with residues of opposite charge revealed that Asp 72 in S2 and Glu 93 in S3 contributed to membrane insertion of S3 and S4, and increased the stability of S4 in the membrane. These results indicate that the SA-I function of S4, unique among K + channels studied to date, promotes the insertion of S3 into the membrane, and that the charged residues essential for voltage sensing contribute to the membrane-insertion of the voltage sensor domain in KvAP. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Differential dynamic and structural behavior of lipid-cholesterol domains in model membranes.

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Luis F Aguilar

Full Text Available Changes in the cholesterol (Chol content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs for cuvette and giant unilamellar vesicles (GUVs for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC and dioctadecyl phosphatidylcholine (DOPC in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones observed in the superficial region of the hydrophilic-hydrophobic interface, and these changes depend on the state of the lamella and (ii the incorporation of cholesterol into the lamella induces an increase in the orientation dynamics in the deep region of the phospholipid acyl chains with a corresponding decrease in the orientation at the region close to the polar lipid headgroups. The microscopy data from DOPC/DPPC/Chol GUVs using Laurdan generalized polarization (Laurdan GP suggest that a high cholesterol content in the bilayer weakens the stability of the water hydrogen bond network and hence the stability of the liquid-ordered phase (Lo.

Nanoscale domain formation of phosphatidylinositol 4-phosphate in the plasma and vacuolar membranes of living yeast cells.

Science.gov (United States)

Tomioku, Kan-Na; Shigekuni, Mikiko; Hayashi, Hiroki; Yoshida, Akane; Futagami, Taiki; Tamaki, Hisanori; Tanabe, Kenji; Fujita, Akikazu

2018-05-01

In budding yeast Saccharomyces cerevisiae, PtdIns(4)P serves as an essential signalling molecule in the Golgi complex, endosomal system, and plasma membrane, where it is involved in the control of multiple cellular functions via direct interactions with PtdIns(4)P-binding proteins. To analyse the distribution of PtdIns(4)P in yeast cells at a nanoscale level, we employed an electron microscopy technique that specifically labels PtdIns(4)P on the freeze-fracture replica of the yeast membrane. This method minimizes the possibility of artificial perturbation, because molecules in the membrane are physically immobilised in situ. We observed that PtdIns(4)P is localised on the cytoplasmic leaflet, but not the exoplasmic leaflet, of the plasma membrane, Golgi body, vacuole, and vesicular structure membranes. PtdIns(4)P labelling was not observed in the membrane of the endoplasmic reticulum, and in the outer and inner membranes of the nuclear envelope or mitochondria. PtdIns(4)P forms clusters of plasma membrane and vacuolar membrane according to point pattern analysis of immunogold labelling. There are three kinds of compartments in the cytoplasmic leaflet of the plasma membrane. In the present study, we showed that PtdIns(4)P is specifically localised in the flat undifferentiated plasma membrane compartment. In the vacuolar membrane, PtdIns(4)P was concentrated in intramembrane particle (IMP)-deficient raft-like domains, which are tightly bound to lipid droplets, but not surrounding IMP-rich non-raft domains in geometrical IMP-distributed patterns in the stationary phase. This is the first report showing microdomain formations of PtdIns(4)P in the plasma membrane and vacuolar membrane of budding yeast cells at a nanoscale level, which will illuminate the functionality of PtdIns(4)P in each membrane. Copyright © 2018 Elsevier GmbH. All rights reserved.

Domain 4 (D4) of Perfringolysin O to Visualize Cholesterol in Cellular Membranes-The Update.

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Maekawa, Masashi

2017-03-03

The cellular membrane of eukaryotes consists of phospholipids, sphingolipids, cholesterol and membrane proteins. Among them, cholesterol is crucial for various cellular events (e.g., signaling, viral/bacterial infection, and membrane trafficking) in addition to its essential role as an ingredient of steroid hormones, vitamin D, and bile acids. From a micro-perspective, at the plasma membrane, recent emerging evidence strongly suggests the existence of lipid nanodomains formed with cholesterol and phospholipids (e.g., sphingomyelin, phosphatidylserine). Thus, it is important to elucidate how cholesterol behaves in membranes and how the behavior of cholesterol is regulated at the molecular level. To elucidate the complexed characteristics of cholesterol in cellular membranes, a couple of useful biosensors that enable us to visualize cholesterol in cellular membranes have been recently developed by utilizing domain 4 (D4) of Perfringolysin O (PFO, theta toxin), a cholesterol-binding toxin. This review highlights the current progress on development of novel cholesterol biosensors that uncover new insights of cholesterol in cellular membranes.

Detergent/nanodisc screening for high-resolution NMR studies of an integral membrane protein containing a cytoplasmic domain.

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Christos Tzitzilonis

Full Text Available Because membrane proteins need to be extracted from their natural environment and reconstituted in artificial milieus for the 3D structure determination by X-ray crystallography or NMR, the search for membrane mimetic that conserve the native structure and functional activities remains challenging. We demonstrate here a detergent/nanodisc screening study by NMR of the bacterial α-helical membrane protein YgaP containing a cytoplasmic rhodanese domain. The analysis of 2D [(15N,(1H]-TROSY spectra shows that only a careful usage of low amounts of mixed detergents did not perturb the cytoplasmic domain while solubilizing in parallel the transmembrane segments with good spectral quality. In contrast, the incorporation of YgaP into nanodiscs appeared to be straightforward and yielded a surprisingly high quality [(15N,(1H]-TROSY spectrum opening an avenue for the structural studies of a helical membrane protein in a bilayer system by solution state NMR.

Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C ₂ A domain in asynchronous neurotransmitter release

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Voleti, Rashmi; Tomchick, Diana R.; Südhof, Thomas C.; Rizo, Josep

2017-09-18

Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturating conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of L-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.

FlnA binding to PACSIN2 F-BAR domain regulates membrane tubulation in megakaryocytes and platelets.

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Begonja, Antonija Jurak; Pluthero, Fred G; Suphamungmee, Worawit; Giannini, Silvia; Christensen, Hilary; Leung, Richard; Lo, Richard W; Nakamura, Fumihiko; Lehman, William; Plomann, Markus; Hoffmeister, Karin M; Kahr, Walter H A; Hartwig, John H; Falet, Hervé

2015-07-02

Bin-Amphiphysin-Rvs (BAR) and Fes-CIP4 homology BAR (F-BAR) proteins generate tubular membrane invaginations reminiscent of the megakaryocyte (MK) demarcation membrane system (DMS), which provides membranes necessary for future platelets. The F-BAR protein PACSIN2 is one of the most abundant BAR/F-BAR proteins in platelets and the only one reported to interact with the cytoskeletal and scaffold protein filamin A (FlnA), an essential regulator of platelet formation and function. The FlnA-PACSIN2 interaction was therefore investigated in MKs and platelets. PACSIN2 associated with FlnA in human platelets. The interaction required FlnA immunoglobulin-like repeat 20 and the tip of PACSIN2 F-BAR domain and enhanced PACSIN2 F-BAR domain membrane tubulation in vitro. Most human and wild-type mouse platelets had 1 to 2 distinct PACSIN2 foci associated with cell membrane GPIbα, whereas Flna-null platelets had 0 to 4 or more foci. Endogenous PACSIN2 and transfected enhanced green fluorescent protein-PACSIN2 were concentrated in midstage wild-type mouse MKs in a well-defined invagination of the plasma membrane reminiscent of the initiating DMS and dispersed in the absence of FlnA binding. The DMS appeared less well defined, and platelet territories were not readily visualized in Flna-null MKs. We conclude that the FlnA-PACSIN2 interaction regulates membrane tubulation in MKs and platelets and likely contributes to DMS formation. © 2015 by The American Society of Hematology.

Interactions between lipids and proteins are critical for organization of plasma membrane-ordered domains in tobacco BY-2 cells.

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Grosjean, Kevin; Der, Christophe; Robert, Franck; Thomas, Dominique; Mongrand, Sébastien; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

2018-06-27

The laterally heterogeneous plant plasma membrane (PM) is organized into finely controlled specialized areas that include membrane-ordered domains. Recently, the spatial distribution of such domains within the PM has been identified as playing a key role in cell responses to environmental challenges. To examine membrane order at a local level, BY-2 tobacco suspension cell PMs were labelled with an environment-sensitive probe (di-4-ANEPPDHQ). Four experimental models were compared to identify mechanisms and cell components involved in short-term (1 h) maintenance of the ordered domain organization in steady-state cell PMs: modulation of the cytoskeleton or the cell wall integrity of tobacco BY-2 cells; and formation of giant vesicles using either a lipid mixture of tobacco BY-2 cell PMs or the original lipid and protein combinations of the tobacco BY-2 cell PM. Whilst inhibiting phosphorylation or disrupting either the cytoskeleton or the cell wall had no observable effects, we found that lipids and proteins significantly modified both the abundance and spatial distribution of ordered domains. This indicates the involvement of intrinsic membrane components in the local physical state of the plant PM. Our findings support a major role for the 'lipid raft' model, defined as the sterol-dependent ordered assemblies of specific lipids and proteins in plant PM organization.

Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

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Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

2015-12-29

The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

The GRP1 PH domain, like the AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P(3).

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Pilling, Carissa; Landgraf, Kyle E; Falke, Joseph J

2011-11-15

During the appearance of the signaling lipid PI(3,4,5)P(3), an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P(3)-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P(2) and bind the rare PI(3,4,5)P(3) target lipid with sufficiently high affinity. Our previous study of the E17K mutant of the protein kinase B (AKT1) PH domain, together with evidence from Carpten et al. [Carpten, J. D., et al. (2007) Nature 448, 439-444], revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P(2), thereby playing an essential role in specific PI(3,4,5)P(3) targeting [Landgraf, K. E., et al. (2008) Biochemistry 47, 12260-12269]. The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P(3)-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P(2) affinity and constitutive plasma membrane targeting. To test this hypothesis, we investigated the E345 residue, a putative sentry glutamate, of the general receptor for phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into the GRP1 PH domain enhances PI(4,5)P(2) affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in the AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P(2) releases the E345K GRP1 PH domain into the cytoplasm, and the efficiency of this release increases when Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K [Carpten, J. D., et al. (2007) Nature 448, 439-444; Lindhurst, M. J., et al

Transcription factor Nrf1 is topologically repartitioned across membranes to enable target gene transactivation through its acidic glucose-responsive domains.

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Zhang, Yiguo; Ren, Yonggang; Li, Shaojun; Hayes, John D

2014-01-01

The membrane-bound Nrf1 transcription factor regulates critical homeostatic and developmental genes. The conserved N-terminal homology box 1 (NHB1) sequence in Nrf1 targets the cap'n'collar (CNC) basic basic-region leucine zipper (bZIP) factor to the endoplasmic reticulum (ER), but it is unknown how its activity is controlled topologically within membranes. Herein, we report a hitherto unknown mechanism by which the transactivation activity of Nrf1 is controlled through its membrane-topology. Thus after Nrf1 is anchored within ER membranes, its acidic transactivation domains (TADs), including the Asn/Ser/Thr-rich (NST) glycodomain situated between acidic domain 1 (AD1) and AD2, are transiently translocated into the lumen of the ER, where NST is glycosylated in the presence of glucose to yield an inactive 120-kDa Nrf1 glycoprotein. Subsequently, portions of the TADs partially repartition across membranes into the cyto/nucleoplasmic compartments, whereupon an active 95-kDa form of Nrf1 accumulates, a process that is more obvious in glucose-deprived cells and may involve deglycosylation. The repartitioning of Nrf1 out of membranes is monitored within this protein by its acidic-hydrophobic amphipathic glucose-responsive domains, particularly the Neh5L subdomain within AD1. Therefore, the membrane-topological organization of Nrf1 dictates its post-translational modifications (i.e. glycosylation, the putative deglycosylation and selective proteolysis), which together control its ability to transactivate target genes.

Transport mechanisms in capillary condensation of water at a single-asperity nanoscopic contact.

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Sirghi, Lucel

2012-02-07

Transport mechanisms involved in capillary condensation of water menisci in nanoscopic gaps between hydrophilic surfaces are investigated theoretically and experimentally by atomic force microscopy (AFM) measurements of capillary force. The measurements showed an instantaneous formation of a water meniscus by coalescence of the water layers adsorbed on the AFM tip and sample surfaces, followed by a time evolution of meniscus toward a stationary state corresponding to thermodynamic equilibrium. This dynamics of the water meniscus is indicated by time evolution of the meniscus force, which increases with the contact time toward its equilibrium value. Two water transport mechanisms competing in this meniscus dynamics are considered: (1) Knudsen diffusion and condensation of water molecules in the nanoscopic gap and (2) adsorption of water molecules on the surface region around the contact and flow of the surface water toward the meniscus. For the case of very hydrophilic surfaces, the dominant role of surface water transportation on the meniscus dynamics is supported by the results of the AFM measurements of capillary force of water menisci formed at sliding tip-sample contacts. These measurements revealed that fast movement of the contact impedes on the formation of menisci at thermodynamic equilibrium because the flow of the surface water is too slow to reach the moving meniscus.

Growth of apatite on chitosan-multiwall carbon nanotube composite membranes

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Yang Jun; Yao Zhiwen [State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, No 14, 3rd Section South People' s Road, Chengdu 610041 (China); Tang Changyu [Department of Polymer Science and Materials, Sichuan University (China); Darvell, B.W. [Dental Materials Science, Faculty of Dentistry, University of Hong Kong (Hong Kong); Zhang Hualin; Pan Lingzhan; Liu Jingsong [State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, No 14, 3rd Section South People' s Road, Chengdu 610041 (China); Chen Zhiqing, E-mail: yangj0710@gmail.com [State Key Laboratory of Oral Diseases, West China College of Stomatology, Sichuan University, No 14, 3rd Section South People' s Road, Chengdu 610041 (China)

2009-07-30

Bioactive membranes for guided tissue regeneration would be of value for periodontal therapy. Chitosan-multiwall carbon nanotube (CS-MWNT) composites were treated to deposit nanoscopic apatite for MWNT proportions of 0-4 mass%. Fourier-transform infrared spectroscopy, scanning electron microscopy, energy-dispersive X-ray analysis, and X-ray diffraction were used for characterization. Apatite was formed on the CS-MWNT composites at low MWNT concentrations, but the dispersion of the MWNT affects the crystallite size and the Ca/P molar ratio of the composite. The smallest crystallite size was 9 nm at 1 mass% MWNT.

NMR Determination of Protein Partitioning into Membrane Domains with Different Curvatures and Application to the Influenza M2 Peptide

Science.gov (United States)

Wang, Tuo; Cady, Sarah D.; Hong, Mei

2012-01-01

The M2 protein of the influenza A virus acts both as a drug-sensitive proton channel and mediates virus budding through membrane scission. The segment responsible for causing membrane curvature is an amphipathic helix in the cytoplasmic domain of the protein. Here, we use 31P and 13C solid-state NMR to examine M2-induced membrane curvature. M2(22–46), which includes only the transmembrane (TM) helix, and M2(21–61), which contains an additional amphipathic helix, are studied. 31P chemical shift lineshapes indicate that M2(21–61) causes a high-curvature isotropic phase to both cholesterol-rich virus-mimetic membranes and 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers, whereas M2(22–46) has minimal effect. The lamellar and isotropic domains have distinct 31P isotropic chemical shifts, indicating perturbation of the lipid headgroup conformation by the amphipathic helix. 31P- and 13C-detected 1H T2 relaxation and two-dimensional peptide-lipid correlation spectra show that M2(21–61) preferentially binds to the high-curvature domain. 31P linewidths indicate that the isotropic vesicles induced by M2(21–61) are 10–35 nm in diameter, and the virus-mimetic vesicles are smaller than the 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles. A strong correlation is found between high membrane curvature and weak drug-binding ability of the TM helix. Thus, the M2 amphipathic helix causes membrane curvature, which in turn perturbs the TM helix conformation, abolishing drug binding. These NMR experiments are applicable to other curvature-inducing membrane proteins such as fusion proteins and antimicrobial peptides. PMID:22385849

Contribution of Adsorbed Protein Films to Nanoscopic Vibrations Exhibited by Bacteria Adhering through Ligand-Receptor Bonds.

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Song, Lei; Sjollema, Jelmer; Norde, Willem; Busscher, Henk J; van der Mei, Henny C

2015-09-29

Bacteria adhering to surfaces exhibit nanoscopic vibrations that depend on the viscoelasticity of the bond. The quantification of the nanoscopic vibrations of bacteria adhering to surfaces provides new opportunities to better understand the properties of the bond through which bacteria adhere and the mechanisms by which they resist detachment. Often, however, bacteria do not adhere to bare surfaces but to adsorbed protein films, on which adhesion involves highly specific ligand-receptor binding next to nonspecific DLVO interaction forces. Here we determine the contribution of adsorbed salivary protein and fibronectin films to vibrations exhibited by adhering streptococci and staphylococci, respectively. The streptococcal strain used has the ability to adhere to adsorbed salivary proteins films through antigen I/II ligand-receptor binding, while the staphylococcal strain used adheres to adsorbed fibronectin films through a proteinaceous ligand-receptor bond. In the absence of ligand-receptor binding, electrostatic interactions had a large impact on vibration amplitudes of adhering bacteria on glass. On an adsorbed salivary protein film, vibration amplitudes of adhering streptococci depended on the film softness as determined by QCM-D and were reduced after film fixation using glutaraldehyde. On a relatively stiff fibronectin film, cross-linking the film in glutaraldehyde hardly reduced its softness, and accordingly fibronectin film softness did not contribute to vibration amplitudes of adhering staphylococci. However, fixation of the staphylococcus-fibronectin bond further decreased vibration amplitudes, while fixation of the streptococcus bond hardly impacted vibration amplitudes. Summarizing, this study shows that both the softness of adsorbed protein films and the properties of the bond between an adhering bacterium and an adsorbed protein film play an important role in bacterial vibration amplitudes. These nanoscopic vibrations reflect the viscoelasticity of the

Nanoscopic X-ray fluorescence imaging and quantification of intracellular key-elements in cryofrozen Friedreich's ataxia fibroblasts.

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Björn De Samber

Full Text Available Synchrotron radiation based nanoscopic X-ray fluorescence (SR nano-XRF analysis can visualize trace level elemental distribution in a fully quantitative manner within single cells. However, in-air XRF analysis requires chemical fixation modifying the cell's chemical composition. Here, we describe first nanoscopic XRF analysis upon cryogenically frozen (-150°C fibroblasts at the ID16A-NI 'Nano-imaging' end-station located at the European Synchrotron Radiation Facility (ESRF in Grenoble (France. Fibroblast cells were obtained from skin biopsies from control and Friedreich's ataxia (FRDA patients. FRDA is an autosomal recessive disorder with dysregulation of iron metabolism as a key feature. By means of the X-ray Fundamental Parameter (FP method, including absorption correction of the ice layer deposited onto the fibroblasts, background-corrected mass fraction elemental maps of P, S, Cl, K, Ca, Fe and Zn of entire cryofrozen human fibroblasts were obtained. Despite the presence of diffracting microcrystals in the vitreous ice matrix and minor sample radiation damage effects, clusters of iron-rich hot-spots with similar mass fractions were found in the cytoplasm of both control and FRDA fibroblasts. Interestingly, no significant difference in the mean iron concentration was found in the cytoplasm of FRDA fibroblasts, but a significant decrease in zinc concentration. This finding might underscore metal dysregulation, beyond iron, in cells derived from FRDA patients. In conclusion, although currently having slightly increased limits of detection (LODs compared to non-cryogenic mode, SR based nanoscopic XRF under cryogenic sample conditions largely obliterates the debate on chemical sample preservation and provides a unique tool for trace level elemental imaging in single cells close to their native state with a superior spatial resolution of 20 nm.

Cholesterol trafficking and raft-like membrane domain composition mediate scavenger receptor class B type 1-dependent lipid sensing in intestinal epithelial cells.

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Morel, Etienne; Ghezzal, Sara; Lucchi, Géraldine; Truntzer, Caroline; Pais de Barros, Jean-Paul; Simon-Plas, Françoise; Demignot, Sylvie; Mineo, Chieko; Shaul, Philip W; Leturque, Armelle; Rousset, Monique; Carrière, Véronique

2018-02-01

Scavenger receptor Class B type 1 (SR-B1) is a lipid transporter and sensor. In intestinal epithelial cells, SR-B1-dependent lipid sensing is associated with SR-B1 recruitment in raft-like/ detergent-resistant membrane domains and interaction of its C-terminal transmembrane domain with plasma membrane cholesterol. To clarify the initiating events occurring during lipid sensing by SR-B1, we analyzed cholesterol trafficking and raft-like domain composition in intestinal epithelial cells expressing wild-type SR-B1 or the mutated form SR-B1-Q445A, defective in membrane cholesterol binding and signal initiation. These features of SR-B1 were found to influence both apical cholesterol efflux and intracellular cholesterol trafficking from plasma membrane to lipid droplets, and the lipid composition of raft-like domains. Lipidomic analysis revealed likely participation of d18:0/16:0 sphingomyelin and 16:0/0:0 lysophosphatidylethanolamine in lipid sensing by SR-B1. Proteomic analysis identified proteins, whose abundance changed in raft-like domains during lipid sensing, and these included molecules linked to lipid raft dynamics and signal transduction. These findings provide new insights into the role of SR-B1 in cellular cholesterol homeostasis and suggest molecular links between SR-B1-dependent lipid sensing and cell cholesterol and lipid droplet dynamics. Copyright © 2017 Elsevier B.V. All rights reserved.

Axonal Membranes and Their Domains: Assembly and Function of the Axon Initial Segment and Node of Ranvier

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Andrew D. Nelson

2017-05-01

Full Text Available Neurons are highly specialized cells of the nervous system that receive, process and transmit electrical signals critical for normal brain function. Here, we review the intricate organization of axonal membrane domains that facilitate rapid action potential conduction underlying communication between complex neuronal circuits. Two critical excitable domains of vertebrate axons are the axon initial segment (AIS and the nodes of Ranvier, which are characterized by the high concentrations of voltage-gated ion channels, cell adhesion molecules and specialized cytoskeletal networks. The AIS is located at the proximal region of the axon and serves as the site of action potential initiation, while nodes of Ranvier, gaps between adjacent myelin sheaths, allow rapid propagation of the action potential through saltatory conduction. The AIS and nodes of Ranvier are assembled by ankyrins, spectrins and their associated binding partners through the clustering of membrane proteins and connection to the underlying cytoskeleton network. Although the AIS and nodes of Ranvier share similar protein composition, their mechanisms of assembly are strikingly different. Here we will cover the mechanisms of formation and maintenance of these axonal excitable membrane domains, specifically highlighting the similarities and differences between them. We will also discuss recent advances in super resolution fluorescence imaging which have elucidated the arrangement of the submembranous axonal cytoskeleton revealing a surprising structural organization necessary to maintain axonal organization and function. Finally, human mutations in axonal domain components have been associated with a growing number of neurological disorders including severe cognitive dysfunction, epilepsy, autism, neurodegenerative diseases and psychiatric disorders. Overall, this review highlights the assembly, maintenance and function of axonal excitable domains, particularly the AIS and nodes of

Graphene Quantum Dot-Aerogel: From Nanoscopic to Macroscopic Fluorescent Materials. Sensing Polyaromatic Compounds in Water.

Science.gov (United States)

Martín-Pacheco, Ana; Del Río Castillo, Antonio Esaú; Martín, Cristina; Herrero, María Antonia; Merino, Sonia; García Fierro, José Luis; Díez-Barra, Enrique; Vázquez, Ester

2018-05-17

Fluorescence based on quantum confinement is a property restricted to the nanoscopic range. The incorporation of nanoparticles in a three-dimensional polymeric network could afford macroscopic scaffolds that show nanoscopic properties. Moreover, if these scaffolds are based on strong bonds, the stability of the resulting materials can be preserved, thus enhancing their final applications. We report for the first time the preparation of a graphene quantum dot (GQD) composite based on a cationic covalent network. This new material has unusual features: (i) the final composite remains stable after several swelling-deswelling cycles, thus demonstrating strong interactions between GQDs and the polymeric material, and therefore it could be used as a portable system. (ii) Fluorescence emission in the composite and in solution is quasi-independent to the excitation wavelength. (iii) However, and in contrast to the behavior observed in GQD solutions, the fluorescence of the composite remains unaltered over a wide pH range and in the presence of different ions commonly found in tap water. (iv) Fluorescence quenching is only observed as a consequence of molecules that bear aromatic systems, and this could be applied to the preparation of in situ water sensors.

Functional activity of Gi alpha protein in detergent resistant membrane domains from rat brain cortex

Czech Academy of Sciences Publication Activity Database

Stöhr, Jiří; Rudajev, Vladimír; Bouřová, Lenka; Lisý, Václav; Novotný, Jiří; Svoboda, Petr

2007-01-01

Roč. 101, Suppl.1 (2007), s. 52-52 ISSN 0022-3042. [European Society for Neurochemistry Meeting /17./. 19.05.2007-22.05.2007, Salamanca] Institutional research plan: CEZ:AV0Z50110509 Keywords : cpo1 * GABAB receptor * Gi protein * membrane domains Subject RIV: ED - Physiology

Structure and function of the Juxta membrane domain of the human epidermal growth factor receptor by NMR spectroscopy

International Nuclear Information System (INIS)

Choowongkomon, Kiattawee; Carlin, Cathleen; Sonnichsen, Frank D.

2005-10-01

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family involved in the regulation of cellular proliferation and differentiation. Its juxta membrane domain (JX), the region located between the transmembrane and kinase domains, plays important roles in receptor trafficking since both basolateral sorting in polarized epithelial cells and lysosomal sorting signals are identified in this region. In order to understand the regulation of these signals, we characterized the structural properties of recombinant JX domain in dodecyl phosphocholine detergent (DPC) by nuclear magnetic resonance (NMR) spectroscopy. In DPC micelles, structures derived from NMR data showed three amphipathic, helical segments. Two equivalent average structural models on the surface of micelles were obtained that differ only in the relative orientation between the first and second helices. Our data suggests that the activity of sorting signals may be regulated by their membrane association and restricted accessibility in the intact receptor

Arabidopsis dynamin-related protein 1E in sphingolipid-enriched plasma membrane domains is associated with the development of freezing tolerance.

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Minami, Anzu; Tominaga, Yoko; Furuto, Akari; Kondo, Mariko; Kawamura, Yukio; Uemura, Matsuo

2015-08-01

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin-related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol-enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye-labelled plasma membrane, providing evidence that DRP1E localizes non-uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol-enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Dynamic shaping of cellular membranes by phospholipids and membrane-deforming proteins.

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Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

2014-10-01

All cellular compartments are separated from the external environment by a membrane, which consists of a lipid bilayer. Subcellular structures, including clathrin-coated pits, caveolae, filopodia, lamellipodia, podosomes, and other intracellular membrane systems, are molded into their specific submicron-scale shapes through various mechanisms. Cells construct their micro-structures on plasma membrane and execute vital functions for life, such as cell migration, cell division, endocytosis, exocytosis, and cytoskeletal regulation. The plasma membrane, rich in anionic phospholipids, utilizes the electrostatic nature of the lipids, specifically the phosphoinositides, to form interactions with cytosolic proteins. These cytosolic proteins have three modes of interaction: 1) electrostatic interaction through unstructured polycationic regions, 2) through structured phosphoinositide-specific binding domains, and 3) through structured domains that bind the membrane without specificity for particular phospholipid. Among the structured domains, there are several that have membrane-deforming activity, which is essential for the formation of concave or convex membrane curvature. These domains include the amphipathic helix, which deforms the membrane by hemi-insertion of the helix with both hydrophobic and electrostatic interactions, and/or the BAR domain superfamily, known to use their positively charged, curved structural surface to deform membranes. Below the membrane, actin filaments support the micro-structures through interactions with several BAR proteins as well as other scaffold proteins, resulting in outward and inward membrane micro-structure formation. Here, we describe the characteristics of phospholipids, and the mechanisms utilized by phosphoinositides to regulate cellular events. We then summarize the precise mechanisms underlying the construction of membrane micro-structures and their involvements in physiological and pathological processes. Copyright © 2014 the

Short length transmembrane domains having voluminous exoplasmic halves determine retention of Type II membrane proteins in the Golgi complex

OpenAIRE

Quiroga, Rodrigo; Trenchi, Alejandra; Gonzalez Montoro, Ayelén; Valdez, Javier Esteban; Maccioni, Hugo Jose Fernando

2017-01-01

It is still unclear why some proteins that travel along the secretory pathway are retained in the Golgi complex whereas others make their way to the plasma membrane. Recent bioinformatic analyses on a large number of single-spanning membrane proteins support the hypothesis that specific features of the transmembrane domain (TMD) are relevant to the sorting of these proteins to particular organelles. Here we experimentally test this hypothesis for Golgi and plasma membrane proteins. Using the ...

Factors Determining the Oxygen Permeability of Biological Membranes: Oxygen Transport Across Eye Lens Fiber-Cell Plasma Membranes.

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Subczynski, Witold Karol; Widomska, Justyna; Mainali, Laxman

2017-01-01

Electron paramagnetic resonance (EPR) spin-label oximetry allows the oxygen permeability coefficient to be evaluated across homogeneous lipid bilayer membranes and, in some cases, across coexisting membrane domains without their physical separation. The most pronounced effect on oxygen permeability is observed for cholesterol, which additionally induces the formation of membrane domains. In intact biological membranes, integral proteins induce the formation of boundary and trapped lipid domains with a low oxygen permeability. The effective oxygen permeability coefficient across the intact biological membrane is affected not only by the oxygen permeability coefficients evaluated for each lipid domain but also by the surface area occupied by these domains in the membrane. All these factors observed in fiber cell plasma membranes of clear human eye lenses are reviewed here.

Crystallization and preliminary crystallographic studies of the C-terminal domain of outer membrane protein A from enterohaemorrhagic Escherichia coli

International Nuclear Information System (INIS)

Gu, Jiang; Ji, Xiaowei; Qi, Jianxun; Ma, Ying; Mao, Xuhu; Zou, Quanming

2010-01-01

In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. Outer membrane protein A (OmpA) of enterohaemorrhagic Escherichia coli (EHEC) plays multiple roles in bacterial physiology and pathogenesis, such as mediation of bacterial conjunction, maintenance of cell shape, induction of adhesion of EHEC to host cells etc. Better understanding of the functions of OmpA will help in the control of EHEC infections. OmpA is composed of two domains: the N-terminal domain and the C-terminal domain. The N-terminal domain is a β-barrel structure and embeds in the outer membrane of the bacterium. The structure and function of the C-terminal domain of OmpA (OmpAC) remain elusive. In this study, recombinant OmpAC from EHEC was purified and crystallized and a diffraction data set was collected to 2.7 Å resolution. The crystals belonged to space group I4 1 32, with unit-cell parameter a = 158.99 Å. The Matthews coefficient and solvent content were calculated to be 2.55 Å 3 Da −1 and 51.77%, respectively, for two molecules in the asymmetric unit

Delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains

Czech Academy of Sciences Publication Activity Database

Bouřová, Lenka; Koštrnová, Alexandra; Hejnová, Lucie; Moravcová, Zuzana; Moon, H. E.; Novotný, Jiří; Milligan, G.; Svoboda, Petr

2003-01-01

Roč. 85, č. 1 (2003), s. 34-49 ISSN 0022-3042 R&D Projects: GA MŠk LN00A026; GA ČR GA309/01/0255 Grant - others:Welcome Trust(GB) xx Institutional research plan: CEZ:AV0Z5011922 Keywords : membrane domains * GTPase activity * G protein coupling Subject RIV: CE - Biochemistry Impact factor: 4.825, year: 2003

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

International Nuclear Information System (INIS)

Hu Chuan; Hardee, Deborah; Minnear, Fred

2007-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Yu, Qianlong [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Blissard, Gary W. [Boyce Thompson Institute, Cornell University, Ithaca, NY 14853, United State (United States); Liu, Tong-Xian [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China); Li, Zhaofei, E-mail: zhaofeili73@outlook.com [State Key Laboratory of Crop Stress Biology for Arid Areas, Key Laboratory of Northwest Loess Plateau Crop Pest Management of Ministry of Agriculture, College of Plant Protection, Northwest A& F University, Yangling, Shaanxi 712100 (China)

2016-01-15

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

Immunochemical and autoantigenic properties of the globular domain of basement membrane collagen (type IV).

Science.gov (United States)

von der Mark, H; Oberbäumer, I; Timpl, R; Kemler, R; Wick, G

1985-02-01

Polyclonal rabbit antibodies raised against the globular domain NC1 of collagen IV from human placenta and a mouse tumor react with conformational antigenic determinants present on the NC1 hexamers and also with the three major subunits obtained after dissociation. The antibodies recognized unique structures within basement membranes and showed a broad tissue reactivity but only limited species cross-reactivity. Using these antibodies, it was possible to detect small amounts of collagen IV antigens from cell cultures and in serum. Monoclonal rat antibodies against mouse NC1 revealed a similar reaction potential. Autoantibodies could be produced in mice against mouse NC1 which react with kidney and lung basement membranes in a pathological manner, mimicking Goodpasture syndrome.

Accessibility Changes within Diphtheria Toxin T Domain upon Membrane Penetration Probed by Hydrogen Exchange and Mass Spectrometry

Czech Academy of Sciences Publication Activity Database

Man, Petr; Montagner, C.; Vitrac, H.; Kavan, Daniel; Pichard, S.; Gillet, D.; Forest, E.; Forge, V.

2011-01-01

Roč. 414, č. 1 (2011), s. 123-134 ISSN 0022-2836 Institutional research plan: CEZ:AV0Z50200510 Keywords : diphtheria toxin * translocation domain * protein/membrane interactions Subject RIV: CE - Biochemistry Impact factor: 4.001, year: 2011

Peptidoglycan-associated outer membrane protein Mep45 of rumen anaerobe Selenomonas ruminantium forms a non-specific diffusion pore via its C-terminal transmembrane domain.

Science.gov (United States)

Kojima, Seiji; Hayashi, Kanako; Tochigi, Saeko; Kusano, Tomonobu; Kaneko, Jun; Kamio, Yoshiyuki

2016-10-01

The major outer membrane protein Mep45 of Selenomonas ruminantium, an anaerobic Gram-negative bacterium, comprises two distinct domains: the N-terminal S-layer homologous (SLH) domain that protrudes into the periplasm and binds to peptidoglycan, and the remaining C-terminal transmembrane domain, whose function has been unknown. Here, we solubilized and purified Mep45 and characterized its function using proteoliposomes reconstituted with Mep45. We found that Mep45 forms a nonspecific diffusion channel via its C-terminal region. The channel was permeable to solutes smaller than a molecular weight of roughly 600, and the estimated pore radius was 0.58 nm. Truncation of the SLH domain did not affect the channel property. On the basis of the fact that Mep45 is the most abundant outer membrane protein in S. ruminantium, we conclude that Mep45 serves as a main pathway through which small solutes diffuse across the outer membrane of this bacterium.

Giant plasma membrane vesicles: models for understanding membrane organization.

Science.gov (United States)

Levental, Kandice R; Levental, Ilya

2015-01-01

The organization of eukaryotic membranes into functional domains continues to fascinate and puzzle cell biologists and biophysicists. The lipid raft hypothesis proposes that collective lipid interactions compartmentalize the membrane into coexisting liquid domains that are central to membrane physiology. This hypothesis has proven controversial because such structures cannot be directly visualized in live cells by light microscopy. The recent observations of liquid-liquid phase separation in biological membranes are an important validation of the raft hypothesis and enable application of the experimental toolbox of membrane physics to a biologically complex phase-separated membrane. This review addresses the role of giant plasma membrane vesicles (GPMVs) in refining the raft hypothesis and expands on the application of GPMVs as an experimental model to answer some of key outstanding problems in membrane biology. Copyright © 2015 Elsevier Inc. All rights reserved.

Studying Mechanosensitivity of Two-Pore Domain K+ Channels in Cellular and Reconstituted Proteoliposome Membranes.

Science.gov (United States)

Del Mármol, Josefina; Rietmeijer, Robert A; Brohawn, Stephen G

2018-01-01

Mechanical force sensation is fundamental to a wide breadth of biology from the classic senses of touch, pain, hearing, and balance to less conspicuous sensations of proprioception, blood pressure, and osmolarity and basic aspects of cell growth, differentiation, and development. These diverse and essential systems use force-gated (or mechanosensitive) ion channels that convert mechanical stimuli into cellular electrical signals. TRAAK, TREK1, and TREK2 are K + -selective ion channels of the two-pore domain K + (K2P) family that are mechanosensitive: they are gated open by increasing membrane tension. TRAAK and TREK channels are thought to play roles in somatosensory and other mechanosensory processes in neuronal and non-neuronal tissues. Here, we present protocols for three assays to study mechanical activation of these channels in cell membranes: (1) cell swelling, (2) cell poking, and (3) patched membrane stretching. Patched membrane stretching is also applicable to the study of mechanosensitive K2P channel activity in a cell-free system and a procedure for proteoliposome reconstitution and patching is also presented. These approaches are also readily applicable to the study of other mechanosensitive ion channels.

Deciphering the BAR code of membrane modulators.

Science.gov (United States)

Salzer, Ulrich; Kostan, Julius; Djinović-Carugo, Kristina

2017-07-01

The BAR domain is the eponymous domain of the "BAR-domain protein superfamily", a large and diverse set of mostly multi-domain proteins that play eminent roles at the membrane cytoskeleton interface. BAR domain homodimers are the functional units that peripherally associate with lipid membranes and are involved in membrane sculpting activities. Differences in their intrinsic curvatures and lipid-binding properties account for a large variety in membrane modulating properties. Membrane activities of BAR domains are further modified and regulated by intramolecular or inter-subunit domains, by intermolecular protein interactions, and by posttranslational modifications. Rather than providing detailed cell biological information on single members of this superfamily, this review focuses on biochemical, biophysical, and structural aspects and on recent findings that paradigmatically promote our understanding of processes driven and modulated by BAR domains.

Overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of the periplasmic domain of outer membrane protein A from Acinetobacter baumannii

International Nuclear Information System (INIS)

Park, Jeong Soon; Lee, Woo Cheol; Choi, Saehae; Yeo, Kwon Joo; Song, Jung Hyun; Han, Young-Hyun; Lee, Je Chul; Kim, Seung Il; Jeon, Young Ho; Cheong, Chaejoon; Kim, Hye-Yeon

2011-01-01

The crystallization of the OmpA periplasmic domain from A. baumannii is described. Outer membrane protein A from Acinetobacter baumannii (AbOmpA) is a major outer membrane protein and a key player in the bacterial pathogenesis that induces host cell death. AbOmpA is presumed to consist of an N-terminal β-barrel transmembrane domain and a C-terminal periplasmic OmpA-like domain. In this study, the recombinant C-terminal periplasmic domain of AbOmpA was overexpressed in Escherichia coli, purified and crystallized using the vapour-diffusion method. A native diffraction data set was collected to a resolution of 2.0 Å using synchrotron radiation. The space group of the crystal was P2 1 , with unit-cell parameters a = 58.24, b = 98.59, c = 97.96 Å, β = 105.92°. The native crystal contained seven or eight molecules per asymmetric unit and had a calculated Matthews coefficient of 2.93 or 2.56 Å 3 Da −1

Membrane dynamics

DEFF Research Database (Denmark)

Bendix, Pól Martin

2015-01-01

Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

APPL proteins FRET at the BAR: direct observation of APPL1 and APPL2 BAR domain-mediated interactions on cell membranes using FRET microscopy.

Directory of Open Access Journals (Sweden)

Heidi J Chial

2010-08-01

Full Text Available Human APPL1 and APPL2 are homologous RAB5 effectors whose binding partners include a diverse set of transmembrane receptors, signaling proteins, and phosphoinositides. APPL proteins associate dynamically with endosomal membranes and are proposed to function in endosome-mediated signaling pathways linking the cell surface to the cell nucleus. APPL proteins contain an N-terminal Bin/Amphiphysin/Rvs (BAR domain, a central pleckstrin homology (PH domain, and a C-terminal phosphotyrosine binding (PTB domain. Previous structural and biochemical studies have shown that the APPL BAR domains mediate homotypic and heterotypic APPL-APPL interactions and that the APPL1 BAR domain forms crescent-shaped dimers. Although previous studies have shown that APPL minimal BAR domains associate with curved cell membranes, direct interaction between APPL BAR domains on cell membranes in vivo has not been reported.Herein, we used a laser-scanning confocal microscope equipped with a spectral detector to carry out fluorescence resonance energy transfer (FRET experiments with cyan fluorescent protein/yellow fluorescent protein (CFP/YFP FRET donor/acceptor pairs to examine interactions between APPL minimal BAR domains at the subcellular level. This comprehensive approach enabled us to evaluate FRET levels in a single cell using three methods: sensitized emission, standard acceptor photobleaching, and sequential acceptor photobleaching. We also analyzed emission spectra to address an outstanding controversy regarding the use of CFP donor/YFP acceptor pairs in FRET acceptor photobleaching experiments, based on reports that photobleaching of YFP converts it into a CFP-like species.All three methods consistently showed significant FRET between APPL minimal BAR domain FRET pairs, indicating that they interact directly in a homotypic (i.e., APPL1-APPL1 and APPL2-APPL2 and heterotypic (i.e., APPL1-APPL2 manner on curved cell membranes. Furthermore, the results of our experiments

The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

Science.gov (United States)

Naito, Daisuke; Ogata, Takehiro; Hamaoka, Tetsuro; Nakanishi, Naohiko; Miyagawa, Kotaro; Maruyama, Naoki; Kasahara, Takeru; Taniguchi, Takuya; Nishi, Masahiro; Matoba, Satoaki; Ueyama, Tomomi

2015-12-15

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function. Copyright © 2015 the American Physiological Society.

Sequential, ordered acquisition of antibodies to Plasmodium falciparum erythrocyte membrane protein 1 domains

DEFF Research Database (Denmark)

Cham, Gerald K K; Turner, Louise; Lusingu, John

2009-01-01

The binding of erythrocytes infected with mature blood stage parasites to the vascular bed is key to the pathogenesis of malignant malaria. The binding is mediated by members of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1s can be divided into groups, and it has pr....... The identification of PfEMP1 domains expressed by parasites causing disease in infants and young children is important for development of vaccines protecting against severe malaria.......The binding of erythrocytes infected with mature blood stage parasites to the vascular bed is key to the pathogenesis of malignant malaria. The binding is mediated by members of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. PfEMP1s can be divided into groups, and it has...... previously been suggested that parasites expressing group A or B/A PfEMP1s are most pathogenic. To test the hypothesis that the first malaria infections in infants and young children are dominated by parasites expressing A and B/A PfEMP1s, we measured the plasma Ab level against 48 recombinant PfEMP1 domains...

The influence of nanoscopically thin silver films on bacterial viability and attachment.

Science.gov (United States)

Ivanova, Elena P; Hasan, Jafar; Truong, Vi Khanh; Wang, James Y; Raveggi, Massimo; Fluke, Christopher; Crawford, Russell J

2011-08-01

The physicochemical and bactericidal properties of thin silver films have been analysed. Silver films of 3 and 150 nm thicknesses were fabricated using a magnetron sputtering thin-film deposition system. X-ray photoelectron and energy dispersive X-ray spectroscopy and atomic force microscopy analyses confirmed that the resulting surfaces were homogeneous, and that silver was the most abundant element present on both surfaces, being 45 and 53 at.% on the 3- and 150-nm films, respectively. Inductively coupled plasma time of flight mass spectroscopy (ICP-TOF-MS) was used to measure the concentration of silver ions released from these films. Concentrations of 0.9 and 5.2 ppb were detected for the 3- and 150-nm films, respectively. The surface wettability of the films remained nearly identical for both film thicknesses, displaying a static water contact angle of 95°, while the surface free energy of the 150-nm film was found to be slightly greater than that of the 3-nm film, being 28.8 and 23.9 mN m(-1), respectively. The two silver film thicknesses exhibited statistically significant differences in surface topographic profiles on the nanoscopic scale, with R (a), R (q) and R (max) values of 1.4, 1.8 and 15.4 nm for the 3-nm film and 0.8, 1.2 and 10.7 nm for the 150-nm film over a 5 × 5 μm scanning area. Confocal scanning laser microscopy and scanning electron microscopy revealed that the bactericidal activity of the 3-nm silver film was not significant, whereas the nanoscopically smoother 150-nm silver film exhibited appreciable bactericidal activity towards Pseudomonas aeruginosa ATCC 9027 cells and Staphylococcus aureus CIP 65.8 cells, obtaining up to 75% and 27% sterilisation effect, respectively.

Hierarchical, domain type-specific acquisition of antibodies to Plasmodium falciparum erythrocyte membrane protein 1 in Tanzanian children

DEFF Research Database (Denmark)

Cham, Gerald K K; Turner, Louise; Kurtis, Jonathan D

2010-01-01

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of malaria-infected erythrocytes. PfEMP1 attaches to the vascular lining and allows infected erythrocytes to avoid filtration through the spleen. Each parasite genome encodes about 60 diffe...... and play a major role in limiting parasite multiplication in the blood.......Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a variant antigen expressed on the surface of malaria-infected erythrocytes. PfEMP1 attaches to the vascular lining and allows infected erythrocytes to avoid filtration through the spleen. Each parasite genome encodes about 60...... different PfEMP1 variants, each PfEMP1 comprises several domains in its extracellular region, and the PfEMP1 repertoire in different parasites contains domain types that are serologically cross-reactive. In this longitudinal study, we followed 672 children living in an area of high malaria transmission...

The Antitumor Effect of Single-domain Antibodies Directed Towards Membrane-associated Catalase and Superoxide Dismutase.

Science.gov (United States)

Bauer, Georg; Motz, Manfred

2016-11-01

Neutralizing single-domain antibodies directed towards catalase or superoxide dismutase (SOD) caused efficient reactivation of intercellular reactive oxygen species/reactive nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling specifically in human tumor cells. Single-domain antibodies targeted tumor cell-specific membrane-associated SOD and catalase, but not the corresponding intracellular enzymes. They were shown to be about 200-fold more effective than corresponding classical recombinant antigen-binding fragments and more than four log steps more efficient than monoclonal antibodies. Combined addition of single-domain antibodies against catalase and SOD caused a remarkable synergistic effect. Proof-of-concept experiments in immunocompromised mice using human tumor xenografts and single-domain antibodies directed towards SOD showed an inhibition of tumor growth. Neutralizing single-domain antibodies directed to catalase and SOD also caused a very strong synergistic effect with the established chemotherapeutic agent taxol, indicating an overlap of signaling pathways. This effect might also be useful in order to avoid unwanted side-effects and to drastically lower the costs for taxol-based therapy. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Far-field interaction of focused relativistic electron beams in electron energy loss spectroscopy of nanoscopic platelets

OpenAIRE

Itskovsky, M. A.; Cohen, H.; Maniv, T.

2008-01-01

A quantum mechanical scattering theory for relativistic, highly focused electron beams near nanoscopic platelets is presented, revealing a new excitation mechanism due to the electron wave scattering from the platelet edges. Radiative electromagnetic excitations within the light cone are shown to arise, allowed by the breakdown of momentum conservation along the beam axis in the inelastic scattering process. Calculated for metallic (silver and gold) and insulating (SiO2 and MgO) nanoplatelets...

Texture of lipid bilayer domains

DEFF Research Database (Denmark)

Jensen, Uffe Bernchou; Brewer, Jonathan R.; Midtiby, Henrik Skov

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 well studied, the possibility of texture in gel domains has so far not been examined. When using polarized light for two-photon excitation of the fluorescent lipid probe Laurdan, the emission intensity is highly sensitive to the angle between the polarization and the tilt orientation of lipid acyl...... 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...

Nanoscopic surfactant behavior of the porin MspA in aqueous media

Directory of Open Access Journals (Sweden)

Ayomi S. Perera

2013-04-01

Full Text Available The mycobacterial porin MspA is one of the most stable channel proteins known to date. MspA forms vesicles at low concentrations in aqueous buffers. Evidence from dynamic light scattering, transmission electron microscopy and zeta-potential measurements by electrophoretic light scattering indicate that MspA behaves like a nanoscale surfactant. The extreme thermostability of MspA allows these investigations to be carried out at temperatures as high as 343 K, at which most other proteins would quickly denature. The principles of vesicle formation of MspA as a function of temperature and the underlying thermodynamic factors are discussed here. The results obtained provide crucial evidence in support of the hypothesis that, during vesicle formation, nanoscopic surfactant molecules, such as MspA, deviate from the principles underlined in classical surface chemistry.

The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter

OpenAIRE

Modali, Sita D.; Zgurskaya, Helen I.

2011-01-01

Escherichia coli MacAB-TolC is a tri-partite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation o...

Cholesterol Bilayer Domains in the Eye Lens Health: A Review.

Science.gov (United States)

Widomska, Justyna; Subczynski, Witold K; Mainali, Laxman; Raguz, Marija

2017-12-01

The most unique biochemical characteristic of the eye lens fiber cell plasma membrane is its extremely high cholesterol content, the need for which is still unclear. It is evident, however, that the disturbance of Chol homeostasis may result in damages associated with cataracts. Electron paramagnetic resonance methods allow discrimination of two types of lipid domains in model membranes overloaded with Chol, namely, phospholipid-cholesterol domains and pure Chol bilayer domains. These domains are also detected in human lens lipid membranes prepared from the total lipids extracted from lens cortices and nuclei of donors from different age groups. Independent of the age-related changes in phospholipid composition, the physical properties of phospholipid-Chol domains remain the same for all age groups and are practically identical for cortical and nuclear membranes. The presence of Chol bilayer domains in these membranes provides a buffering capacity for cholesterol concentration in the surrounding phospholipid-Chol domains, keeping it at a constant saturating level and thus keeping the physical properties of the membrane consistent with and independent of changes in phospholipid composition. It seems that the presence of Chol bilayer domains plays an integral role in the regulation of cholesterol-dependent processes in fiber cell plasm membranes and in the maintenance of fiber cell membrane homeostasis.

The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter.

Science.gov (United States)

Modali, Sita D; Zgurskaya, Helen I

2011-08-01

Escherichia coli MacAB-TolC is a tripartite macrolide efflux transporter driven by hydrolysis of ATP. In this complex, MacA is the periplasmic membrane fusion protein that stimulates the activity of MacB transporter and establishes the link with the outer membrane channel TolC. The molecular mechanism by which MacA stimulates MacB remains unknown. Here, we report that the periplasmic membrane proximal domain of MacA plays a critical role in functional MacA-MacB interactions and stimulation of MacB ATPase activity. Binding of MacA to MacB stabilizes the ATP-bound conformation of MacB, whereas interactions with both MacB and TolC affect the conformation of MacA. A single G353A substitution in the C-terminus of MacA inactivates MacAB-TolC function by changing the conformation of the membrane proximal domain of MacA and disrupting the proper assembly of the MacA-MacB complex. We propose that MacA acts in transport by promoting MacB transition into the closed ATP-bound conformation and in this respect, is similar to the periplasmic solute-binding proteins. © 2011 Blackwell Publishing Ltd.

Molecular mechanisms of protein-cholesterol interactions in plasma membranes: Functional distinction between topological (tilted) and consensus (CARC/CRAC) domains.

Science.gov (United States)

Fantini, Jacques; Di Scala, Coralie; Baier, Carlos J; Barrantes, Francisco J

2016-09-01

The molecular mechanisms that control the multiple possible modes of protein association with membrane cholesterol are remarkably convergent. These mechanisms, which include hydrogen bonding, CH-π stacking and dispersion forces, are used by a wide variety of extracellular proteins (e.g. microbial or amyloid) and membrane receptors. Virus fusion peptides penetrate the membrane of host cells with a tilted orientation that is compatible with a transient interaction with cholesterol; this tilted orientation is also characteristic of the process of insertion of amyloid proteins that subsequently form oligomeric pores in the plasma membrane of brain cells. Membrane receptors that are associated with cholesterol generally display linear consensus binding motifs (CARC and CRAC) characterized by a triad of basic (Lys/Arg), aromatic (Tyr/phe) and aliphatic (Leu/Val) amino acid residues. In some cases, the presence of both CARC and CRAC within the same membrane-spanning domain allows the simultaneous binding of two cholesterol molecules, one in each membrane leaflet. In this review the molecular basis and the functional significance of the different modes of protein-cholesterol interactions in plasma membranes are discussed. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Different domains of Bacillus thuringiensis delta-endotoxins can bind to insect midgut membrane proteins on ligand blots

NARCIS (Netherlands)

Maagd, de R.A.; Klei, van der H.; Bakker, P.L.; Stiekema, W.J.; Bosch, D.

1996-01-01

We investigated the role of the constituent domains of the CryIA(b) and CryIA(c) δ-endotoxins in binding to midgut epithelial cell membrane proteins of Spodoptera exigua and Manduca sexta on ligand blots. A collection of wild- type and CryIC-CryIA hybrid toxins was used for this purpose. As

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery.

Science.gov (United States)

Poëa-Guyon, Sandrine; Ammar, Mohamed Raafet; Erard, Marie; Amar, Muriel; Moreau, Alexandre W; Fossier, Philippe; Gleize, Vincent; Vitale, Nicolas; Morel, Nicolas

2013-10-28

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

Raft-like membrane domains in pathogenic microorganisms.

Science.gov (United States)

Farnoud, Amir M; Toledo, Alvaro M; Konopka, James B; Del Poeta, Maurizio; London, Erwin

2015-01-01

The lipid bilayer of the plasma membrane is thought to be compartmentalized by the presence of lipid-protein microdomains. In eukaryotic cells, microdomains composed of sterols and sphingolipids, commonly known as lipid rafts, are believed to exist, and reports on the presence of sterol- or protein-mediated microdomains in bacterial cell membranes are also appearing. Despite increasing attention, little is known about microdomains in the plasma membrane of pathogenic microorganisms. This review attempts to provide an overview of the current state of knowledge of lipid rafts in pathogenic fungi and bacteria. The current literature on characterization of microdomains in pathogens is reviewed, and their potential role in growth, pathogenesis, and drug resistance is discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of their pathogenesis and development of raft-mediated approaches for therapy. Copyright © 2015 Elsevier Inc. All rights reserved.

Gene transfer and expression in human neutrophils. The phox homology domain of p47phox translocates to the plasma membrane but not to the membrane of mature phagosomes

Directory of Open Access Journals (Sweden)

Brzezinska Agnieszka A

2006-12-01

Full Text Available Abstract Background Neutrophils are non-dividing cells with poor survival after isolation. Consequently, exogenous gene expression in neutrophils is challenging. We report here the transfection of genes and expression of active proteins in human primary peripheral neutrophils using nucleofection. Results Exogenous gene expression in human neutrophils was achieved 2 h post-transfection. We show that neutrophils transfected by nucleofection are functional cells, able to respond to soluble and particulate stimuli. They conserved the ability to undergo physiological processes including phagocytosis. Using this technique, we were able to show that the phox homology (PX domain of p47phox localizes to the plasma membrane in human neutrophils. We also show that RhoB, but not the PX domain of p47phox, is translocated to the membrane of mature phagosomes. Conclusion We demonstrated that cDNA transfer and expression of exogenous protein in human neutrophils is compatible with cell viability and is no longer a limitation for the study of protein function in human neutrophils.

Biochemical characterization of domain-specific glycoproteins of the rat hepatocyte plasma membrane

International Nuclear Information System (INIS)

Bartles, J.R.; Braiterman, L.T.; Hubbard, A.L.

1985-01-01

Seven integral proteins (CE 9, HA 21, HA 116, HA 16, HA 4, HA 201, and HA 301) were isolated from rat hepatocyte plasma membranes by immunoaffinity chromatography on monoclonal antibody-Sepharose. Six of the proteins (all but HA 16) exhibit domain-specific localizations (either bile canalicular or sinusoidal/lateral) about the hepatocyte surface. The authors identified three of these protein antigens as leucine aminopeptidase (HA 201), dipeptidyl peptidase IV (HA 301), and the asialoglycoprotein receptor (HA 116). They also developed 125 I-lectin blotting procedures that, when used in conjunction with chemical and glycosidase treatments, permitted a comparison of the types of oligosaccharides present on the seven proteins. All seven are sialoglycoproteins, based upon the effects of prior neuraminidase and periodate-aniline-cyanoborohydride treatments of blots on labeling by 125 I-wheat germ agglutinin. Depending upon the protein, they estimated the presence of 2-26 N-linked oligosaccharides/polypeptide chain from the Mr reductions accompanying chemical or enzymatic deglycosylation. Three of these mature plasma membrane proteins (HA 21, HA 116, and HA 4) have both high mannose-type and complex-type oligosaccharides on every copy of their polypeptide chains

Electroformation of Giant Unilamellar Vesicles from Native Membranes and Organic Lipid Mixtures for the Study of Lipid Domains under Physiological Ionic-Strength Conditions

DEFF Research Database (Denmark)

Montes, Ruth; Ahyayauch, Hasna; Ibarguren, Maitane

2010-01-01

Giant unilamellar vesicles (GUVs) constitute a cell-sized model membrane system that allows direct visualization of particular membrane-related phenomena, such as domain formation, at the level of single vesicles using fluorescence microscopy-related techniques. Currently available protocols...... for the preparation of GUVs work only at very low salt concentrations, thus precluding experimentation under physiological conditions. In addition, the GUVs thus obtained lack membrane compositional asymmetry. Here we show how to prepare GUVs using a new protocol based on the electroformation method either from...... native membranes or organic lipid mixtures at physiological ionic strength. Additionally, we describe methods to test whether membrane proteins and glycosphingolipids preserve their natural orientation after electroformation of GUVs composed of native membranes...

Domain-to-domain coupling in voltage-sensing phosphatase.

Science.gov (United States)

Sakata, Souhei; Matsuda, Makoto; Kawanabe, Akira; Okamura, Yasushi

2017-01-01

Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.

Interaction of calmodulin with the calmodulin binding domain of the plasma membrane Ca2+ pump

International Nuclear Information System (INIS)

Vorherr, T.; James, P.; Krebs, J.; Carafoli, E.; McCormick, D.J.; Penniston, J.T.; Enyedi, A.

1990-01-01

Peptides corresponding to the calmodulin binding domain of the plasma membrane Ca 2+ pump were synthesized, and their interaction with calmodulin was studied with circular dichroism, infrared spectroscopy, nuclear magnetic resonance, and fluorescence techniques. They corresponded to the complete calmodulin binding domain (28 residues), to its first 15 or 20 amino acids, and to its C-terminal 14 amino acids. The first three peptides interacted with calmodulin. The K value was similar to that of the intact enzyme in the 28 and 20 amino acid peptides, but increased substantially in the shorter 15 amino acid peptide. The 14 amino acid peptide corresponding to the C-terminal portion of the domain failed to bind calmodulin. 2D NMR experiments on the 20 amino acid peptides have indicated that the interaction occurred with the C-terminal half of calmodulin. A tryptophan that is conserved in most calmodulin binding domains of proteins was replaced by other amino acids, giving rise to modified peptides which had lower affinity for calmodulin. An 18 amino acid peptide corresponding to an acidic sequence immediately N-terminal to the calmodulin binding domain which is likely to be a Ca 2+ binding site in the pump was also synthesized. Circular dichroism experiments have shown that it interacted with calmodulin binding domain, supporting the suggestion that the latter, or a portion of it, may act as a natural inhibitor of the pump

Mapping the membrane-aqueous border for the voltage-sensing domain of a potassium channel.

Science.gov (United States)

Neale, Edward J; Rong, Honglin; Cockcroft, Christopher J; Sivaprasadarao, Asipu

2007-12-28

Voltage-sensing domains (VSDs) play diverse roles in biology. As integral components, they can detect changes in the membrane potential of a cell and couple these changes to activity of ion channels and enzymes. As independent proteins, homologues of the VSD can function as voltage-dependent proton channels. To sense voltage changes, the positively charged fourth transmembrane segment, S4, must move across the energetically unfavorable hydrophobic core of the bilayer, which presents a barrier to movement of both charged species and protons. To reduce the barrier to S4 movement, it has been suggested that aqueous crevices may penetrate the protein, reducing the extent of total movement. To investigate this hypothesis in a system containing fully functional channels in a native environment with an intact membrane potential, we have determined the contour of the membrane-aqueous border of the VSD of KvAP in Escherichia coli by examining the chemical accessibility of introduced cysteines. The results revealed the contour of the membrane-aqueous border of the VSD in its activated conformation. The water-inaccessible regions of S1 and S2 correspond to the standard width of the membrane bilayer (~28 A), but those of S3 and S4 are considerably shorter (> or = 40%), consistent with aqueous crevices pervading both the extracellular and intracellular ends. One face of S3b and the entire S3a were water-accessible, reducing the water-inaccessible region of S3 to just 10 residues, significantly shorter than for S4. The results suggest a key role for S3 in reducing the distance S4 needs to move to elicit gating.

Nanoscopic diffusion studies on III-V compound semiconductor structures: Experiment and theory

Science.gov (United States)

Gonzalez Debs, Mariam

The electronic structure of multilayer semiconductor heterostructures is affected by the detailed compositional profiles throughout the structure and at critical interfaces. The extent of interdiffusion across these interfaces places limits on both the processing time and temperatures for many applications based on the resultant compositional profile and associated electronic structure. Atomic and phenomenological methods were used in this work through the combination of experiment and theory to understand the nanoscopic mechanisms in complex heterostructures. Two principal studies were conducted. Tin diffusion in GaAs was studied by fitting complex experimental diffusion profiles to a phenomenological model which involved the diffusion of substitutional and interstitial dopant atoms. A methodology was developed combining both the atomistic model and the use of key features within these experimentally-obtained diffusion profiles to determine meaningful values of the transport and defect reaction rate parameters. Interdiffusion across AlSb/GaSb multi-quantum well interfaces was also studied. The chemical diffusion coefficient characterizing the AlSb/GaSb diffusion couple was quantitatively determined by fitting the observed photoluminescence (PL) peak shifts to the solution of the Schrodinger equation using a potential derived from the solution of the diffusion equation to quantify the interband transition energy shifts. First-principles calculations implementing Density Functional Theory were performed to study the thermochemistry of point defects as a function of local environment, allowing a direct comparison of interfacial and bulk diffusion phenomena within these nanoscopic structures. Significant differences were observed in the Ga and Al vacancy formation energies at the AlSb/GaSb interface when compared to bulk AlSb and GaSb with the largest change found for Al vacancies. The AlSb/GaSb structures were further studied using positron annihilation spectroscopy

Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases

DEFF Research Database (Denmark)

Larsen, Jannik Bruun; Jensen, Martin Borch; Bhatia, Vikram Kjøller

2015-01-01

Trafficking and sorting of membrane-anchored Ras GTPases are regulated by partitioning between distinct membrane domains. Here, in vitro experiments and microscopic molecular theory reveal membrane curvature as a new modulator of N-Ras lipid anchor and palmitoyl chain partitioning. Membrane...

Membrane raft association is a determinant of plasma membrane localization.

Science.gov (United States)

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

2014-06-10

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

Voltage-sensing phosphatase modulation by a C2 domain.

Science.gov (United States)

Castle, Paul M; Zolman, Kevin D; Kohout, Susy C

2015-01-01

The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain, and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate (PIP) lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry (VCF) were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

Improved non-dimensional dynamic influence function method based on tow-domain method for vibration analysis of membranes

Directory of Open Access Journals (Sweden)

SW Kang

2015-02-01

Full Text Available This article introduces an improved non-dimensional dynamic influence function method using a sub-domain method for efficiently extracting the eigenvalues and mode shapes of concave membranes with arbitrary shapes. The non-dimensional dynamic influence function method (non-dimensional dynamic influence function method, which was developed by the authors in 1999, gives highly accurate eigenvalues for membranes, plates, and acoustic cavities, compared with the finite element method. However, it needs the inefficient procedure of calculating the singularity of a system matrix in the frequency range of interest for extracting eigenvalues and mode shapes. To overcome the inefficient procedure, this article proposes a practical approach to make the system matrix equation of the concave membrane of interest into a form of algebraic eigenvalue problem. It is shown by several case studies that the proposed method has a good convergence characteristics and yields very accurate eigenvalues, compared with an exact method and finite element method (ANSYS.

Residues in the membrane-spanning domain core modulate conformation and fusogenicity of the HIV-1 envelope glycoprotein

International Nuclear Information System (INIS)

Shang Liang; Hunter, Eric

2010-01-01

The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant 'core' structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD 'core' do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD 'core' delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusion mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.

Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism ofSphingomyelinase-Induced Hot-Cold Hemolysis

DEFF Research Database (Denmark)

Montes, Ruth; Lopez, David; Sot, Jesus

2008-01-01

Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 °C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 °C. The mechanism of this phenomenon is not understood. PlcHR2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa......) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot-cold hemolysis induced by PlcHR2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR2 activity. After......-cold hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR2 demonstrates the presence of ceramide-rich domains that are rigid at 4 °C but fluid at 37 °C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally...

Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase

International Nuclear Information System (INIS)

Roberts, W.L.; Rosenberry, T.L.

1986-01-01

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase

Long-range interaction between heterogeneously charged membranes.

Science.gov (United States)

Jho, Y S; Brewster, R; Safran, S A; Pincus, P A

2011-04-19

Despite their neutrality, surfaces or membranes with equal amounts of positive and negative charge can exhibit long-range electrostatic interactions if the surface charge is heterogeneous; this can happen when the surface charges form finite-size domain structures. These domains can be formed in lipid membranes where the balance of the different ranges of strong but short-ranged hydrophobic interactions and longer-ranged electrostatic repulsion result in a finite, stable domain size. If the domain size is large enough, oppositely charged domains in two opposing surfaces or membranes can be strongly correlated by the electrostatic interactions; these correlations give rise to an attractive interaction of the two membranes or surfaces over separations on the order of the domain size. We use numerical simulations to demonstrate the existence of strong attractions at separations of tens of nanometers. Large line tensions result in larger domains but also increase the charge density within the domain. This promotes correlations and, as a result, increases the intermembrane attraction. On the other hand, increasing the salt concentration increases both the domain size and degree of domain anticorrelation, but the interactions are ultimately reduced due to increased screening. The result is a decrease in the net attraction as salt concentration is increased. © 2011 American Chemical Society

Sensing voltage across lipid membranes

Science.gov (United States)

Swartz, Kenton J.

2009-01-01

The detection of electrical potentials across lipid bilayers by specialized membrane proteins is required for many fundamental cellular processes such as the generation and propagation of nerve impulses. These membrane proteins possess modular voltage-sensing domains, a notable example being the S1-S4 domains of voltage-activated ion channels. Ground-breaking structural studies on these domains explain how voltage sensors are designed and reveal important interactions with the surrounding lipid membrane. Although further structures are needed to fully understand the conformational changes that occur during voltage sensing, the available data help to frame several key concepts that are fundamental to the mechanism of voltage sensing. PMID:19092925

Novel mechanisms for self-assembled pattern formation in nanoscopic metal films

Science.gov (United States)

Kalyanaraman, R.; Trice, J.; Favazza, C.; Thomas, D.; Sureshkumar, R.

2007-03-01

Classical hydrodynamic theory of dewetting of spinodally unstable thin films (Vrij, Disc. farad. Soc. 1966) predicts a monotonic increase in patterning length scales with increasing film thickness. We verified this effect for nanoscopic Co metal films following melting by ns laser pulses for thickness regime hFavazza et al. Nanotechnology, 2006). However, a dramatic change is observed beyond this thickness hc, with length scales decreasing with increasing h. This novel behavior arises from strong thickness dependence of heating by ultrafast laser light resulting in thermocapillary effects, whose magnitude and sign are thickness dependent. We modified the classical theory, according to which the instability occurs when the stabilizing capillary force is overcome by destabilizing attractive long-range interactions, to include thermocapillary effects. The modified theory accurately predicts the experimentally observed trend. This result suggests that a variety of new length scales can be accessed by robust self-assembly via dewetting of metal films under ultrafast light.

Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

Science.gov (United States)

Imam, Zachary I; Kenyon, Laura E; Carrillo, Adelita; Espinoza, Isai; Nagib, Fatema; Stachowiak, Jeanne C

2016-04-19

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required

1H-detected MAS solid-state NMR experiments enable the simultaneous mapping of rigid and dynamic domains of membrane proteins

Science.gov (United States)

Gopinath, T.; Nelson, Sarah E. D.; Veglia, Gianluigi

2017-12-01

Magic angle spinning (MAS) solid-state NMR (ssNMR) spectroscopy is emerging as a unique method for the atomic resolution structure determination of native membrane proteins in lipid bilayers. Although 13C-detected ssNMR experiments continue to play a major role, recent technological developments have made it possible to carry out 1H-detected experiments, boosting both sensitivity and resolution. Here, we describe a new set of 1H-detected hybrid pulse sequences that combine through-bond and through-space correlation elements into single experiments, enabling the simultaneous detection of rigid and dynamic domains of membrane proteins. As proof-of-principle, we applied these new pulse sequences to the membrane protein phospholamban (PLN) reconstituted in lipid bilayers under moderate MAS conditions. The cross-polarization (CP) based elements enabled the detection of the relatively immobile residues of PLN in the transmembrane domain using through-space correlations; whereas the most dynamic region, which is in equilibrium between folded and unfolded states, was mapped by through-bond INEPT-based elements. These new 1H-detected experiments will enable one to detect not only the most populated (ground) states of biomacromolecules, but also sparsely populated high-energy (excited) states for a complete characterization of protein free energy landscapes.

BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

Directory of Open Access Journals (Sweden)

Kathrin Weber

2013-10-01

Full Text Available BAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a “BCL-XL-addiction” model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models.

Voltage-sensing phosphatase modulation by a C2 domain

Directory of Open Access Journals (Sweden)

Paul M. Castle

2015-04-01

Full Text Available The voltage-sensing phosphatase (VSP is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD, the inter-domain linker, the cytosolic catalytic domain and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5P2 (Kalli et al., 2014. Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in

CX3CL1, a chemokine finely tuned to adhesion: critical roles of the stalk glycosylation and the membrane domain

Directory of Open Access Journals (Sweden)

Mariano A. Ostuni

2014-11-01

Full Text Available The multi-domain CX3CL1 transmembrane chemokine triggers leukocyte adherence without rolling and migration by presenting its chemokine domain (CD to its receptor CX3CR1. Through the combination of functional adhesion assays with structural analysis using FRAP, we investigated the functional role of the other domains of CX3CL1, i.e., its mucin stalk, transmembrane domain, and cytosolic domain. Our results indicate that the CX3CL1 molecular structure is finely adapted to capture CX3CR1 in circulating cells and that each domain has a specific purpose: the mucin stalk is stiffened by its high glycosylation to present the CD away from the membrane, the transmembrane domain generates the permanent aggregation of an adequate amount of monomers to guarantee adhesion and prevent rolling, and the cytosolic domain ensures adhesive robustness by interacting with the cytoskeleton. We propose a model in which quasi-immobile CX3CL1 bundles are organized to quickly generate adhesive patches with sufficiently high strength to capture CX3CR1+ leukocytes but with sufficiently low strength to allow their patrolling behavior.

cDNA cloning of the basement membrane chondroitin sulfate proteoglycan core protein, bamacan: a five domain structure including coiled-coil motifs

DEFF Research Database (Denmark)

Wu, R R; Couchman, J R

1997-01-01

Basement membranes contain several proteoglycans, and those bearing heparan sulfate glycosaminoglycans such as perlecan and agrin usually predominate. Most mammalian basement membranes also contain chondroitin sulfate, and a core protein, bamacan, has been partially characterized. We have now....... The protein sequence has low overall homology, apart from very small NH2- and COOH-terminal motifs. At the junctions between the distal globular domains and the coiled-coil regions lie glycosylation sites, with up to three N-linked oligosaccharides and probably three chondroitin chains. Three other Ser...

Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

DEFF Research Database (Denmark)

Ammendrup-Johnsen, Ina; Thorsen, Thor Seneca; Gether, Ulrik

2012-01-01

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the a...... lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution....

Cholesterol asymmetry in synaptic plasma membranes.

Science.gov (United States)

Wood, W Gibson; Igbavboa, Urule; Müller, Walter E; Eckert, Gunter P

2011-03-01

Lipids are essential for the structural and functional integrity of membranes. Membrane lipids are not randomly distributed but are localized in different domains. A common characteristic of these membrane domains is their association with cholesterol. Lipid rafts and caveolae are examples of cholesterol enriched domains, which have attracted keen interest. However, two other important cholesterol domains are the exofacial and cytofacial leaflets of the plasma membrane. The two leaflets that make up the bilayer differ in their fluidity, electrical charge, lipid distribution, and active sites of certain proteins. The synaptic plasma membrane (SPM) cytofacial leaflet contains over 85% of the total SPM cholesterol as compared with the exofacial leaflet. This asymmetric distribution of cholesterol is not fixed or immobile but can be modified by different conditions in vivo: (i) chronic ethanol consumption; (ii) statins; (iii) aging; and (iv) apoE isoform. Several potential candidates have been proposed as mechanisms involved in regulation of SPM cholesterol asymmetry: apoE, low-density lipoprotein receptor, sterol carrier protein-2, fatty acid binding proteins, polyunsaturated fatty acids, P-glycoprotein and caveolin-1. This review examines cholesterol asymmetry in SPM, potential mechanisms of regulation and impact on membrane structure and function. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Limited cross-reactivity among domains of the Plasmodium falciparum clone 3D7 erythrocyte membrane protein 1 family

DEFF Research Database (Denmark)

Joergensen, Louise; Turner, Louise; Magistrado, Pamela

2006-01-01

The var gene-encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family is responsible for antigenic variation and sequestration of infected erythrocytes during malaria. We have previously grouped the 60 PfEMP1 variants of P. falciparum clone 3D7 into groups A and B/A (category A......) and groups B, B/C, and C (category non-A). Expression of category A molecules is associated with severe malaria, and that of category non-A molecules is associated with uncomplicated malaria and asymptomatic infection. Here we assessed cross-reactivity among 60 different recombinant PfEMP1 domains derived...... from clone 3D7 by using a competition enzyme-linked immunosorbent assay and a pool of plasma from 63 malaria-exposed Tanzanian individuals. We conclude that naturally acquired antibodies are largely directed toward epitopes varying between different domains with a few, mainly category A, domains...

Differential distribution of proteins and lipids in detergent-resistant and detergent-soluble domains in rod outer segment plasma membranes and disks.

Science.gov (United States)

Elliott, Michael H; Nash, Zack A; Takemori, Nobuaki; Fliesler, Steven J; McClellan, Mark E; Naash, Muna I

2008-01-01

Membrane heterogeneity plays a significant role in regulating signal transduction and other cellular activities. We examined the protein and lipid components associated with the detergent-resistant membrane (DRM) fractions from retinal rod outer segment (ROS) disk and plasma membrane-enriched preparations. Proteomics and correlative western blot analysis revealed the presence of alpha and beta subunits of the rod cGMP-gated ion channel and glucose transporter type 1, among other proteins. The glucose transporter was present exclusively in ROS plasma membrane (not disks) and was highly enriched in DRMs, as was the cGMP-gated channel beta-subunit. In contrast, the majority of rod opsin and ATP-binding cassette transporter A4 was localized to detergent-soluble domains in disks. As expected, the cholesterol : fatty acid mole ratio was higher in DRMs than in the corresponding parent membranes (disk and plasma membranes, respectively) and was also higher in disks compared to plasma membranes. Furthermore, the ratio of saturated : polyunsaturated fatty acids was also higher in DRMs compared to their respective parent membranes (disk and plasma membranes). These results confirm that DRMs prepared from both disks and plasma membranes are enriched in cholesterol and in saturated fatty acids compared to their parent membranes. The dominant fatty acids in DRMs were 16 : 0 and 18 : 0; 22 : 6n3 and 18 : 1 levels were threefold higher and twofold lower, respectively, in disk-derived DRMs compared to plasma membrane-derived DRMs. We estimate, based on fatty acid recovery that DRMs account for only approximately 8% of disks and approximately 12% of ROS plasma membrane.

Membrane-tethered peptides patterned after the TRP domain (TRPducins) selectively inhibit TRPV1 channel activity.

Science.gov (United States)

Valente, Pierluigi; Fernández-Carvajal, Asia; Camprubí-Robles, María; Gomis, Ana; Quirce, Susana; Viana, Félix; Fernández-Ballester, Gregorio; González-Ros, José M; Belmonte, Carlos; Planells-Cases, Rosa; Ferrer-Montiel, Antonio

2011-05-01

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC(50)100 μM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.

SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

International Nuclear Information System (INIS)

Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

2009-01-01

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

Dynamics of membrane nanotubes coated with I-BAR

Science.gov (United States)

Barooji, Younes F.; Rørvig-Lund, Andreas; Semsey, Szabolcs; Reihani, S. Nader S.; Bendix, Poul M.

2016-07-01

Membrane deformation is a necessary step in a number of cellular processes such as filopodia and invadopodia formation and has been shown to involve membrane shaping proteins containing membrane binding domains from the IRSp53-MIM protein family. In reconstituted membranes the membrane shaping domains can efficiently deform negatively charged membranes into tubules without any other proteins present. Here, we show that the IM domain (also called I-BAR domain) from the protein ABBA, forms semi-flexible nanotubes protruding into Giant Unilamellar lipid Vesicles (GUVs). By simultaneous quantification of tube intensity and tubular shape we find both the diameter and stiffness of the nanotubes. I-BAR decorated tubes were quantified to have a diameter of ~50 nm and exhibit no stiffening relative to protein free tubes of the same diameter. At high protein density the tubes are immobile whereas at lower density the tubes diffuse freely on the surface of the GUV. Bleaching experiments of the fluorescently tagged I-BAR confirmed that the mobility of the tubes correlates with the mobility of the I-BAR on the GUV membrane. Finally, at low density of I-BAR the protein upconcentrates within tubes protruding into the GUVs. This implies that I-BAR exhibits strong preference for negatively curved membranes.

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains.

Science.gov (United States)

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS.

Modulation of catalytic activity in multi-domain protein tyrosine phosphatases.

Directory of Open Access Journals (Sweden)

Lalima L Madan

Full Text Available Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1 domains, while the membrane-distal (D2 domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A. While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane

OpenAIRE

Doudová, Lenka

2017-01-01

Membrane compartment of Can1 (MCC): specialized functional microdomain of the yeast plasma membrane Yeast plasma membrane is divided into several different compartments. Membrane compartment of Can1 is specific for its protein and lipid composition, furthermore it creates furrow-like invaginations on the plasma membrane. These invaginations are made by multiprotein complexes called eisosomes, which are located in the cytosolic side of MCCs. It was established that this domain plays an importa...

Differential Effect of Plant Lipids on Membrane Organization

Science.gov (United States)

Grosjean, Kevin; Mongrand, Sébastien; Beney, Laurent; Simon-Plas, Françoise; Gerbeau-Pissot, Patricia

2015-01-01

The high diversity of the plant lipid mixture raises the question of their respective involvement in the definition of membrane organization. This is particularly the case for plant plasma membrane, which is enriched in specific lipids, such as free and conjugated forms of phytosterols and typical phytosphingolipids, such as glycosylinositolphosphoceramides. This question was here addressed extensively by characterizing the order level of membrane from vesicles prepared using various plant lipid mixtures and labeled with an environment-sensitive probe. Fluorescence spectroscopy experiments showed that among major phytosterols, campesterol exhibits a stronger ability than β-sitosterol and stigmasterol to order model membranes. Multispectral confocal microscopy, allowing spatial analysis of membrane organization, demonstrated accordingly the strong ability of campesterol to promote ordered domain formation and to organize their spatial distribution at the membrane surface. Conjugated sterol forms, alone and in synergy with free sterols, exhibit a striking ability to order membrane. Plant sphingolipids, particularly glycosylinositolphosphoceramides, enhanced the sterol-induced ordering effect, emphasizing the formation and increasing the size of sterol-dependent ordered domains. Altogether, our results support a differential involvement of free and conjugated phytosterols in the formation of ordered domains and suggest that the diversity of plant lipids, allowing various local combinations of lipid species, could be a major contributor to membrane organization in particular through the formation of sphingolipid-sterol interacting domains. PMID:25575593

Radiative interaction of a focused relativistic electron beam in energy-loss spectroscopy of nanoscopic platelets

International Nuclear Information System (INIS)

Itskovsky, M. A.; Maniv, T.; Cohen, H.

2008-01-01

A quantum-mechanical scattering theory for relativistic, highly focused electron beams in the vacuum near nanoscopic platelets is presented, revealing an excitation mechanism due to the electron wave scattering from the platelet edges. Radiative electromagnetic excitations within the light cone are shown to arise, allowed by the breakdown of momentum conservation along the beam axis in the inelastic-scattering process. Calculated for metallic (silver and gold) and insulating (SiO 2 and MgO) nanoplatelets, radiative features are revealed above the main surface-plasmon-polariton peak, and dramatic enhancements in the electron-energy-loss probability at gaps of the 'classical' spectra are found. The corresponding radiation should be detectable in the vacuum far-field zone, with e beams exploited as sensitive 'tip detectors' of electronically excited nanostructures

Reflectivity and thickness analysis of epiretinal membranes using spectral-domain optical coherence tomography

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Ajay E. Kuriyan

2016-01-01

Full Text Available AIM: To compare thickness and reflectivity spectral domain optical coherence tomography (SD-OCT findings in patients with idiopathic epiretinal membranes (ERMs, before and after ERM peeling surgery, with normal controls. METHODS: A retrospective study analyzed SD-OCTs of eyes with ERMs undergoing ERM peeling surgery by one surgeon from 2008 to 2010 and normal control eyes. SD-OCTs were analyzed using a customized algorithm to measure reflectivity and thickness. The relationship between the SD-OCT findings and best corrected visual acuity (BCVA outcomes was also studied. RESULTS: Thirty-four ERM eyes and 12 normal eyes were identified. Preoperative eyes had high reflectivity and thickness of the group of layers from the internal limiting membrane (ILM to the retinal pigment epithelium (RPE and the group of layers from the ILM to the external limiting membrane (ELM. The values of reflectivity of these two groups of layers decreased postoperatively, but were still higher than normal eyes. In contrast, preoperative eyes had lower reflectivity of two 10×15 pixel regions of interest (ROIs incorporating: 1 ELM + outer nuclear layer (ONL and 2 photoreceptor layer (PRL + RPE, compared to controls. The values of reflectivity of these ROIs increased postoperatively, but were still lower than normal controls. A larger improvement in BCVA postoperatively was correlated with a greater degree of abnormal preoperative reflectivity and thickness findings. CONCLUSION: Quantitative differences in reflectivity and thickness between preoperative, postoperative, and normal SD-OCTs allow assessment of changes in the retina secondary to ERM. Our study identified hyperreflective inner retina changes and hyporeflective outer retina changes in patients with ERMs. SD-OCT quantitative measures of reflectivity and/or thickness of specific groups of retinal layers and/or ROIs correlate with improvement in BCVA.

Visualizing Macular Structures During Membrane Peeling Surgery With an Intraoperative Spectral-Domain Optical Coherence Tomography Device.

Science.gov (United States)

Leisser, Christoph; Hackl, Christoph; Hirnschall, Nino; Luft, Nikolaus; Döller, Birgit; Draschl, Petra; Rigal, Karl; Findl, Oliver

2016-04-01

The aim of this study was to examine the quality of intraoperative visualization of the posterior hyaloid, epiretinal membrane (ERM), inner limiting membrane (ILM), and hyporeflective subfoveal zone with a commercially available, microscope-integrated spectral-domain OCT setup (mi-SD-OCT) (Rescan 700; Carl Zeiss Meditec AG, Germany). Twenty patients prospectively scheduled for pars plana vitrectomy with membrane peeling due to an idiopathic ERM were included. Standard 23-gauge, three-port pars plana vitrectomy with membrane peeling and staining of the ERM with a trypan blue-based chromovitrectomy dye was performed in all cases. Intraoperative SD-OCT was performed before and after peeling and visualization of the posterior hyaloid, ERM, ILM, and presence of subfoveal hyporeflective zones were examined. OCT follow-ups were performed 2 days and 3 months after surgery. The study was approved by the local ethics committee of the city of Vienna. Successful intraoperative visualization of ERM by mi-SD-OCT was possible in all cases. The posterior hyaloid and ILM could not be seen in the mi-SD-OCT scans, whereas an intraoperative subfoveal hyporeflective zone presented in 35% of cases. In 12.5% an independent subfoveal hyporeflective zone presented postoperatively. Visual acuity improved in 93.8% of patients after surgery. mi-SD-OCT appears to be a valuable tool for intraoperative visualization of the ERM and offers immediate visualization of retinal anatomy during peeling. Therefore, it adds to the understanding of intraoperative traumatic changes due to the peeling procedure. Copyright 2016, SLACK Incorporated.

Discovery of novel membrane binding structures and functions

Science.gov (United States)

Kufareva, Irina; Lenoir, Marc; Dancea, Felician; Sridhar, Pooja; Raush, Eugene; Bissig, Christin; Gruenberg, Jean; Abagyan, Ruben; Overduin, Michael

2014-01-01

The function of a protein is determined by its intrinsic activity in the context of its subcellular distribution. Membranes localize proteins within cellular compartments and govern their specific activities. Discovering such membrane-protein interactions is important for understanding biological mechanisms, and could uncover novel sites for therapeutic intervention. Here we present a method for detecting membrane interactive proteins and their exposed residues that insert into lipid bilayers. Although the development process involved analysis of how C1b, C2, ENTH, FYVE, Gla, pleckstrin homology (PH) and PX domains bind membranes, the resulting Membrane Optimal Docking Area (MODA) method yields predictions for a given protein of known three dimensional structures without referring to canonical membrane-targeting modules. This approach was tested on the Arf1 GTPase, ATF2 acetyltransferase, von Willebrand factor A3 domain and Neisseria gonorrhoeae MsrB protein, and further refined with membrane interactive and non-interactive FAPP1 and PKD1 pleckstrin homology domains, respectively. Furthermore we demonstrate how this tool can be used to discover unprecedented membrane binding functions as illustrated by the Bro1 domain of Alix, which was revealed to recognize lysobisphosphatidic acid (LBPA). Validation of novel membrane-protein interactions relies on other techniques such as nuclear magnetic resonance spectroscopy (NMR) which was used here to map the sites of micelle interaction. Together this indicates that genome-wide identification of known and novel membrane interactive proteins and sites is now feasible, and provides a new tool for functional annotation of the proteome. PMID:25394204

Assembly and misassembly of cystic fibrosis transmembrane conductance regulator: folding defects caused by deletion of F508 occur before and after the calnexin-dependent association of membrane spanning domain (MSD) 1 and MSD2.

Science.gov (United States)

Rosser, Meredith F N; Grove, Diane E; Chen, Liling; Cyr, Douglas M

2008-11-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl(-) channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRDeltaF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.

Different functional modes of BAR domain proteins in formation and plasticity of mammalian postsynapses.

Science.gov (United States)

Kessels, Michael M; Qualmann, Britta

2015-09-01

A plethora of cell biological processes involve modulations of cellular membranes. By using extended lipid-binding interfaces, some proteins have the power to shape membranes by attaching to them. Among such membrane shapers, the superfamily of Bin-Amphiphysin-Rvs (BAR) domain proteins has recently taken center stage. Extensive structural work on BAR domains has revealed a common curved fold that can serve as an extended membrane-binding interface to modulate membrane topologies and has allowed the grouping of the BAR domain superfamily into subfamilies with structurally slightly distinct BAR domain subtypes (N-BAR, BAR, F-BAR and I-BAR). Most BAR superfamily members are expressed in the mammalian nervous system. Neurons are elaborately shaped and highly compartmentalized cells. Therefore, analyses of synapse formation and of postsynaptic reorganization processes (synaptic plasticity) - a basis for learning and memory formation - has unveiled important physiological functions of BAR domain superfamily members. These recent advances, furthermore, have revealed that the functions of BAR domain proteins include different aspects. These functions are influenced by the often complex domain organization of BAR domain proteins. In this Commentary, we review these recent insights and propose to classify BAR domain protein functions into (1) membrane shaping, (2) physical integration, (3) action through signaling components, and (4) suppression of other BAR domain functions. © 2015. Published by The Company of Biologists Ltd.

Importance of the hexagonal lipid phase in biological membrane organisation

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Juliette eJouhet

2013-12-01

Full Text Available Abstract:Domains are present in every natural membrane. They are characterised by a distinctive protein and/or lipid composition. Their size is highly variable from the nano- to the micrometer scale. The domains confer specific properties to the membrane leading to original structure and function. The determinants leading to domain organisation are therefore important but remain obscure. This review presents how the ability of lipids to organize into hexagonal II or lamellar phases can promote particular local structures within membranes. Since biological membranes are composed of a mixture of lipids, each with distinctive biophysical properties, lateral and transversal sorting of lipids can promote creation of domains inside the membrane through local modulation of the lipid phase. Lipid biophysical properties have been characterized for long based on in vitro analyses using non-natural lipid molecules; their re-examinations using natural lipids might open interesting perspectives on membrane architecture occurring in vivo in various cellular and physiological contexts.

Importance of the hexagonal lipid phase in biological membrane organization.

Science.gov (United States)

Jouhet, Juliette

2013-01-01

Domains are present in every natural membrane. They are characterized by a distinctive protein and/or lipid composition. Their size is highly variable from the nano- to the micrometer scale. The domains confer specific properties to the membrane leading to original structure and function. The determinants leading to domain organization are therefore important but remain obscure. This review presents how the ability of lipids to organize into hexagonal II or lamellar phases can promote particular local structures within membranes. Since biological membranes are composed of a mixture of lipids, each with distinctive biophysical properties, lateral and transversal sorting of lipids can promote creation of domains inside the membrane through local modulation of the lipid phase. Lipid biophysical properties have been characterized for long based on in vitro analyses using non-natural lipid molecules; their re-examinations using natural lipids might open interesting perspectives on membrane architecture occurring in vivo in various cellular and physiological contexts.

Properties of Fiber Cell Plasma Membranes Isolated from the Cortex and Nucleus of the Porcine Eye Lens

Science.gov (United States)

Mainali, Laxman; Raguz, Marija; O’Brien, William J.; Subczynski, Witold K.

2012-01-01

The organization and physical properties of the lipid bilayer portion of intact cortical and nuclear fiber cell plasma membranes isolated from the eyes lenses of two-year-old pigs were studied using electron paramagnetic resonance (EPR) spin-labeling. Membrane fluidity, hydrophobicity, and the oxygen transport parameter (OTP) were assessed from the EPR spectra of precisely positioned spin labels. Intact cortical and nuclear membranes, which include membrane proteins, were found to contain three distinct lipid environments. These lipid environments were termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain (lipids in protein aggregates). The amount of boundary and trapped lipids was greater in intact nuclear membranes than in cortical membranes. The properties of intact membranes were compared with the organization and properties of lens lipid membranes made of the total lipid extracts from the lens cortex or nucleus. In cortical lens lipid membranes, only one homogenous environment was detected, which was designated as a bulk lipid domain (phospholipid bilayer saturated with cholesterol). Lens lipid membranes prepared from the lens nucleus possessed two domains, assigned as a bulk lipid domain and a cholesterol bilayer domain (CBD). In intact nuclear membranes, it was difficult to discriminate the CBD, which was clearly detected in nuclear lens lipid membranes because the OTP measured in the CBD is the same as in the domain formed by trapped lipids. The two domains unique to intact membranes—namely, the domain formed by boundary lipids and the domain formed by trapped lipids—were most likely formed due to the presence of membrane proteins. It is concluded that formation of rigid and practically impermeable domains is enhanced in the lens nucleus, indicating changes in membrane composition that may help to maintain low oxygen concentration in this lens region. PMID:22326289

Quantification of Protein-Induced Membrane Remodeling Kinetics In Vitro with Lipid Multilayer Gratings

Science.gov (United States)

Lowry, Troy W.; Hariri, Hanaa; Prommapan, Plengchart; Kusi-Appiah, Aubrey; Vafai, Nicholas; Bienkiewicz, Ewa A.; Van Winkle, David H.; Stagg, Scott M.

2016-01-01

The dynamic self-organization of lipids in biological systems is a highly regulated process that enables the compartmentalization of living systems at micro- and nanoscopic scales. Consequently, quantitative methods for assaying the kinetics of supramolecular remodeling such as vesicle formation from planar lipid bilayers or multilayers are needed to understand cellular self-organization. Here, a new nanotechnology-based method for quantitative measurements of lipid–protein interactions is presented and its suitability for quantifying the membrane binding, inflation, and budding activity of the membrane-remodeling protein Sar1 is demonstrated. Lipid multilayer gratings are printed onto surfaces using nanointaglio and exposed to Sar1, resulting in the inflation of lipid multilayers into unilamellar structures, which can be observed in a label-free manner by monitoring the diffracted light. Local variations in lipid multilayer volume on the surface is used to vary substrate availability in a microarray format. A quantitative model is developed that allows quantification of binding affinity (KD) and kinetics (kon and koff). Importantly, this assay is uniquely capable of quantifying membrane remodeling. Upon Sar1-induced inflation of single bilayers from surface supported multilayers, the semicylindrical grating lines are observed to remodel into semispherical buds when a critical radius of curvature is reached. PMID:26649649

The dimer interface of the membrane type 1 matrix metalloproteinase hemopexin domain: crystal structure and biological functions.

Science.gov (United States)

Tochowicz, Anna; Goettig, Peter; Evans, Richard; Visse, Robert; Shitomi, Yasuyuki; Palmisano, Ralf; Ito, Noriko; Richter, Klaus; Maskos, Klaus; Franke, Daniel; Svergun, Dmitri; Nagase, Hideaki; Bode, Wolfram; Itoh, Yoshifumi

2011-03-04

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.

Efficient subgroup C avian sarcoma and leukosis virus receptor activity requires the IgV domain of the Tvc receptor and proper display on the cell membrane.

Science.gov (United States)

Munguia, Audelia; Federspiel, Mark J

2008-11-01

We recently identified and cloned the receptor for subgroup C avian sarcoma and leukosis viruses [ASLV(C)], i.e., Tvc, a protein most closely related to mammalian butyrophilins, which are members of the immunoglobulin protein family. The extracellular domain of Tvc contains two immunoglobulin-like domains, IgV and IgC, which presumably each contain a disulfide bond important for native function of the protein. In this study, we have begun to identify the functional determinants of Tvc responsible for ASLV(C) receptor activity. We found that the IgV domain of the Tvc receptor is responsible for interacting with the glycoprotein of ASLV(C). Additional experiments demonstrated that a domain was necessary as a spacer between the IgV domain and the membrane-spanning domain for efficient Tvc receptor activity, most likely to orient the IgV domain a proper distance from the cell membrane. The effects on ASLV(C) glycoprotein binding and infection efficiency were also studied by site-directed mutagenesis of the cysteine residues of Tvc as well as conserved amino acid residues of the IgV Tvc domain compared to other IgV domains. In this initial analysis of Tvc determinants important for interacting with ASLV(C) glycoproteins, at least two aromatic amino acid residues in the IgV domain of Tvc, Trp-48 and Tyr-105, were identified as critical for efficient ASLV(C) infection. Interestingly, one or more aromatic amino acid residues have been identified as critical determinants in the other ASLV(A-E) receptors for a proper interaction with ASLV glycoproteins. This suggests that the ASLV glycoproteins may share a common mechanism of receptor interaction with an aromatic residue(s) on the receptor critical for triggering conformational changes in SU that initiate the fusion process required for efficient virus infection.

Pair interaction of bilayer-coated nanoscopic particles

International Nuclear Information System (INIS)

Qi-Yi, Zhang

2009-01-01

The pair interaction between bilayer membrane-coated nanosized particles has been explored by using the self-consistent field (SCF) theory. The bilayer membranes are composed of amphiphilic polymers. For different system parameters, the pair-interaction free energies are obtained. Particular emphasis is placed on the analysis of a sequence of structural transformations of bilayers on spherical particles, which occur during their approaching processes. For different head fractions of amphiphiles, the asymmetrical morphologies between bilayers on two particles and the inverted micellar intermediates have been found in the membrane fusion pathway. These results can benefit the fabrication of vesicles as encapsulation vectors for drug and gene delivery. (condensed matter: structure, thermal and mechanical properties)

Lipid-protein interactions in plasma membranes of fiber cells isolated from the human eye lens.

Science.gov (United States)

Raguz, Marija; Mainali, Laxman; O'Brien, William J; Subczynski, Witold K

2014-03-01

The protein content in human lens membranes is extremely high, increases with age, and is higher in the nucleus as compared with the cortex, which should strongly affect the organization and properties of the lipid bilayer portion of intact membranes. To assess these effects, the intact cortical and nuclear fiber cell plasma membranes isolated from human lenses from 41- to 60-year-old donors were studied using electron paramagnetic resonance spin-labeling methods. Results were compared with those obtained for lens lipid membranes prepared from total lipid extracts from human eyes of the same age group [Mainali, L., Raguz, M., O'Brien, W. J., and Subczynski, W. K. (2013) Biochim. Biophys. Acta]. Differences were considered to be mainly due to the effect of membrane proteins. The lipid-bilayer portions of intact membranes were significantly less fluid than lipid bilayers of lens lipid membranes, prepared without proteins. The intact membranes were found to contain three distinct lipid environments termed the bulk lipid domain, boundary lipid domain, and trapped lipid domain. However, the cholesterol bilayer domain, which was detected in cortical and nuclear lens lipid membranes, was not detected in intact membranes. The relative amounts of bulk and trapped lipids were evaluated. The amount of lipids in domains uniquely formed due to the presence of membrane proteins was greater in nuclear membranes than in cortical membranes. Thus, it is evident that the rigidity of nuclear membranes is greater than that of cortical membranes. Also the permeability coefficients for oxygen measured in domains of nuclear membranes were significantly lower than appropriate coefficients measured in cortical membranes. Relationships between the organization of lipids into lipid domains in fiber cells plasma membranes and the organization of membrane proteins are discussed. Copyright © 2014 Elsevier Ltd. All rights reserved.

Dynamics of membrane nanotubes coated with I-BAR

DEFF Research Database (Denmark)

Farhangibarooji, Younes; Rørvig-Lund, Andreas; Semsey, Szabolcs

2016-01-01

Membrane deformation is a necessary step in a number of cellular processes such as filopodia and invadopodia formation and has been shown to involve membrane shaping proteins containing membrane binding domains from the IRSp53-MIM protein family. In reconstituted membranes the membrane shaping...

Radiative interaction of a focused relativistic electron beam in energy-loss spectroscopy of nanoscopic platelets

Science.gov (United States)

Itskovsky, M. A.; Cohen, H.; Maniv, T.

2008-07-01

A quantum-mechanical scattering theory for relativistic, highly focused electron beams in the vacuum near nanoscopic platelets is presented, revealing an excitation mechanism due to the electron wave scattering from the platelet edges. Radiative electromagnetic excitations within the light cone are shown to arise, allowed by the breakdown of momentum conservation along the beam axis in the inelastic-scattering process. Calculated for metallic (silver and gold) and insulating ( SiO2 and MgO) nanoplatelets, radiative features are revealed above the main surface-plasmon-polariton peak, and dramatic enhancements in the electron-energy-loss probability at gaps of the “classical” spectra are found. The corresponding radiation should be detectable in the vacuum far-field zone, with e beams exploited as sensitive “tip detectors” of electronically excited nanostructures.

Introducing Membrane Charge and Membrane Potential to T Cell Signaling

Directory of Open Access Journals (Sweden)

Yuanqing Ma

2017-11-01

Full Text Available While membrane models now include the heterogeneous distribution of lipids, the impact of membrane charges on regulating the association of proteins with the plasma membrane is often overlooked. Charged lipids are asymmetrically distributed between the two leaflets of the plasma membrane, resulting in the inner leaflet being negatively charged and a surface potential that attracts and binds positively charged ions, proteins, and peptide motifs. These interactions not only create a transmembrane potential but they can also facilitate the formation of charged membrane domains. Here, we reference fields outside of immunology in which consequences of membrane charge are better characterized to highlight important mechanisms. We then focus on T cell receptor (TCR signaling, reviewing the evidence that membrane charges and membrane-associated calcium regulate phosphorylation of the TCR–CD3 complex and discuss how the immunological synapse exhibits distinct patterns of membrane charge distribution. We propose that charged lipids, ions in solution, and transient protein interactions form a dynamic equilibrium during T cell activation.

Role for chlamydial inclusion membrane proteins in inclusion membrane structure and biogenesis.

Directory of Open Access Journals (Sweden)

Jeffrey Mital

Full Text Available The chlamydial inclusion membrane is extensively modified by the insertion of type III secreted effector proteins. These inclusion membrane proteins (Incs are exposed to the cytosol and share a common structural feature of a long, bi-lobed hydrophobic domain but little or no primary amino acid sequence similarity. Based upon secondary structural predictions, over 50 putative inclusion membrane proteins have been identified in Chlamydia trachomatis. Only a limited number of biological functions have been defined and these are not shared between chlamydial species. Here we have ectopically expressed several C. trachomatis Incs in HeLa cells and find that they induce the formation of morphologically distinct membranous vesicular compartments. Formation of these vesicles requires the bi-lobed hydrophobic domain as a minimum. No markers for various cellular organelles were observed in association with these vesicles. Lipid probes were incorporated by the Inc-induced vesicles although the lipids incorporated were dependent upon the specific Inc expressed. Co-expression of Inc pairs indicated that some colocalized in the same vesicle, others partially overlapped, and others did not associate at all. Overall, it appears that Incs may have an intrinsic ability to induce membrane formation and that individual Incs can induce membranous structures with unique properties.

Molecular assemblies and membrane domains in multivesicular endosome dynamics

International Nuclear Information System (INIS)

Falguieres, Thomas; Luyet, Pierre-Philippe; Gruenberg, Jean

2009-01-01

Along the degradation pathway, endosomes exhibit a characteristic multivesicular organization, resulting from the budding of vesicles into the endosomal lumen. After endocytosis and transport to early endosomes, activated signaling receptors are incorporated into these intralumenal vesicles through the action of the ESCRT machinery, a process that contributes to terminate signaling. Then, the vesicles and their protein cargo are further transported towards lysosomes for degradation. Evidence also shows that intralumenal vesicles can undergo 'back-fusion' with the late endosome limiting membrane, a route exploited by some pathogens and presumably followed by proteins and lipids that need to be recycled from within the endosomal lumen. This process depends on the late endosomal lipid lysobisphosphatidic acid and its putative effector Alix/AIP1, and is presumably coupled to the invagination of the endosomal limiting membrane at the molecular level via ESCRT proteins. In this review, we discuss the intra-endosomal transport routes in mammalian cells, and in particular the different mechanisms involved in membrane invagination, vesicle formation and fusion in a space inaccessible to proteins known to control intracellular membrane traffic.

Expression, purification, crystallization and preliminary X-ray analysis of calmodulin in complex with the regulatory domain of the plasma-membrane Ca2+-ATPase ACA8

International Nuclear Information System (INIS)

Tidow, Henning; Hein, Kim L.; Baekgaard, Lone; Palmgren, Michael G.; Nissen, Poul

2010-01-01

Plant plasma-membrane Ca 2+ -ATPase is regulated via binding of calmodulin to its autoinhibitory N-terminal domain. In this study, the expression, purification, crystallization and preliminary X-ray diffraction analysis of this protein complex from A. thaliana are reported. Plasma-membrane Ca 2+ -ATPases (PMCAs) are calcium pumps that expel Ca 2+ from eukaryotic cells to maintain overall Ca 2+ homoeostasis and to provide local control of intracellular Ca 2+ signalling. They are of major physiological importance, with different isoforms being essential, for example, for presynaptic and postsynaptic Ca 2+ regulation in neurons, feedback signalling in the heart and sperm motility. In the resting state, PMCAs are autoinhibited by binding of their C-terminal (in mammals) or N-terminal (in plants) tail to two major intracellular loops. Activation requires the binding of calcium-bound calmodulin (Ca 2+ -CaM) to this tail and a conformational change that displaces the autoinhibitory tail from the catalytic domain. The complex between calmodulin and the regulatory domain of the plasma-membrane Ca 2+ -ATPase ACA8 from Arabidopsis thaliana has been crystallized. The crystals belonged to space group C2, with unit-cell parameters a = 176.8, b = 70.0, c = 69.8 Å, β = 113.2°. A complete data set was collected to 3.0 Å resolution and structure determination is in progress in order to elucidate the mechanism of PMCA activation by calmodulin

How the antimicrobial peptides destroy bacteria cell membrane: Translocations vs. membrane buckling

Science.gov (United States)

Golubovic, Leonardo; Gao, Lianghui; Chen, Licui; Fang, Weihai

2012-02-01

In this study, coarse grained Dissipative Particle Dynamics simulation with implementation of electrostatic interactions is developed in constant pressure and surface tension ensemble to elucidate how the antimicrobial peptide molecules affect bilayer cell membrane structure and kill bacteria. We find that peptides with different chemical-physical properties exhibit different membrane obstructing mechanisms. Peptide molecules can destroy vital functions of the affected bacteria by translocating across their membranes via worm-holes, or by associating with membrane lipids to form hydrophilic cores trapped inside the hydrophobic domain of the membranes. In the latter scenario, the affected membranes are strongly corrugated (buckled) in accord with very recent experimental observations [G. E. Fantner et al., Nat. Nanotech., 5 (2010), pp. 280-285].

Tryptic mapping and membrane topology of the benzodiazepine receptor alpha-subunit

Energy Technology Data Exchange (ETDEWEB)

Lentes, K.U.; Venter, J.C.

1986-05-01

Rat brain membrane benzodiazepine receptors (BZR) were photoaffinity labelled specifically (in presence or absence of 6 ..mu..M clonazepam) with 10 nM /sup 3/H-flunitrazepam (FNZ). Digestion of the FNZ-labelled, membrane-bound BZR with 200 ..mu..g trypsin/mg membrane protein yielded H/sub 2/O-soluble BZR-fragments of molecular mass (M/sub r/) 34, 31, 28, 24, 21, 18, 16, 12, 10 and 7kDa. Because the 34kDa-peptide is the largest fragment containing a FNZ-binding site they conclude that this represents the extracellular domain of the BZR. In the remaining pellet two labelled peptides with M/sub r/ of 44kDa and 28kDa were found that required the use of detergents for their solubilization; they therefore contain the membrane anchoring domain. Digestion of the 0.5% Na-deoxycholate solubilized, intact BZR (M/sub r/ 51kDa) resulted in the same tryptic pattern as the membrane form of the receptor plus two larger fragments of M/sub r/ 45kDa and 40kDa. Arrangement of all tryptic fragments with reference to the FNZ binding site reveals a membrane topology of the BZR alpha-subunit with 67% (34kDa) for the extracellular domain, 21% (11kDa) for the membrane anchoring domain and 12% (6kDa) for a putative cytoplasmic domain. The overlap between some of the labelled fragments suggest that the BZ binding site must be located near the membrane surface of the extracellular domain.

Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

Science.gov (United States)

Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.

2017-10-01

Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

Characterization of nanostructures in the live cell plasma membrane utilizing advanced single molecule fluorescence techniques

International Nuclear Information System (INIS)

Brameshuber, M.

2009-01-01

Unrevealing the detailed structure of the cellular plasma membrane at a nanoscopic length scale is the key for understanding the regulation of various signaling pathways or interaction mechanism. Hypotheses postulate the existence of nanoscopic lipid platforms in the cell membrane which are termed lipid- or membrane rafts. Based on biochemical studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. In this thesis I present an ultra-sensitive fluorescence based method which allows for the first time the direct imaging of single mobile rafts in the live cell plasma membrane. The method senses rafts by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. A special photobleaching protocol was developed and used to reduce the surface density of labeled mobile rafts down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per raft was determined by single molecule brightness analysis. For demonstration, I used the consensus markers Bodipy-GM1, a fluorescent lipid analogue, and glycosylphosphatidyl-inositol-anchored monomeric GFP. For both markers I found cholesterol-dependent association in the plasma membrane of living CHO and Jurkat T cells in the resting state, indicating the presence of mobile, stable rafts hosting these probes. I further characterized these structures by taking cell-to-cell variations under consideration. By comparing Bodipy-GM1 with mGFP-GPI homo-association upon temperature variation, two different states - a non-equilibrated and an equilibrated state - could be identified. I conclude that rafts are loaded non-randomly; the characteristic load is maintained during its lifetime in the plasma membrane of a non-activated cell. Beside these

A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

International Nuclear Information System (INIS)

Rosiere, T.K.; Marrs, J.A.; Bouck, G.B.

1990-01-01

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39

Characterization of the immersion properties of the peripheral membrane anchor of the FATC domain of the kinase "target of rapamycin" by NMR, oriented CD spectroscopy, and MD simulations.

Science.gov (United States)

Sommer, Lisa A M; Janke, J Joel; Bennett, W F Drew; Bürck, Jochen; Ulrich, Anne S; Tieleman, D Peter; Dames, Sonja A

2014-05-08

The multidomain ser/thr kinase "target of rapamycin" (TOR) centrally controls eukaryotic growth and metabolism. The C-terminal FATC domain is important for TOR regulation and was suggested to directly mediate TOR-membrane interactions. Here, we present a detailed characterization of the membrane immersion properties of the oxidized and reduced yeast TOR1 FATC domain (2438-2470 = y1fatc). The immersion depth was characterized by NMR-monitored interaction studies with DPC micelles containing paramagnetically tagged 5- or 16-doxyl stearic acid (5-/16-SASL) and by analyzing the paramagnetic relaxation enhancement (PRE) from Mn(2+) in the solvent. Complementary MD-simulations of micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed. Based on oriented CD (OCD) data, the single α-helix of oxidized/reduced y1fatc has an angle to the membrane normal of ∼30-60°/∼35-65° in neutral and ∼5-35°/∼0-30° in negatively charged bilayers. The presented experimentally well-founded models help to better understand how this redox-sensitive peripheral membrane anchor may be part of a network of protein-protein and protein-membrane interactions regulating TOR localization at different cellular membranes. Moreover, the presented work provides a good methodological reference for the structural characterization of other peripherally membrane associating proteins.

MIT domain of Vps4 is a Ca2+-dependent phosphoinositide-binding domain.

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Iwaya, Naoko; Takasu, Hirotoshi; Goda, Natsuko; Shirakawa, Masahiro; Tanaka, Toshiki; Hamada, Daizo; Hiroaki, Hidekazu

2013-05-01

The microtubule interacting and trafficking (MIT) domain is a small protein module that is conserved in proteins of diverged function, such as Vps4, spastin and sorting nexin 15 (SNX15). The molecular function of the MIT domain is protein-protein interaction, in which the domain recognizes peptides containing MIT-interacting motifs. Recently, we identified an evolutionarily related domain, 'variant' MIT domain at the N-terminal region of the microtubule severing enzyme katanin p60. We found that the domain was responsible for binding to microtubules and Ca(2+). Here, we have examined whether the authentic MIT domains also bind Ca(2+). We found that the loop between the first and second α-helices of the MIT domain binds a Ca(2+) ion. Furthermore, the MIT domains derived from Vps4b and SNX15a showed phosphoinositide-binding activities in a Ca(2+)-dependent manner. We propose that the MIT domain is a novel membrane-associating domain involved in endosomal trafficking.

Insight into Phosphatidylinositol-Dependent Membrane Localization of the Innate Immune Adaptor Protein Toll/Interleukin 1 Receptor Domain-Containing Adaptor Protein

Directory of Open Access Journals (Sweden)

Mahesh Chandra Patra

2018-01-01

Full Text Available The toll/interleukin 1 receptor (TIR domain-containing adaptor protein (TIRAP plays an important role in the toll-like receptor (TLR 2, TLR4, TLR7, and TLR9 signaling pathways. TIRAP anchors to phosphatidylinositol (PI 4,5-bisphosphate (PIP2 on the plasma membrane and PI (3,4,5-trisphosphate (PIP3 on the endosomal membrane and assists in recruitment of the myeloid differentiation primary response 88 protein to activated TLRs. To date, the structure and mechanism of TIRAP’s membrane association are only partially understood. Here, we modeled an all-residue TIRAP dimer using homology modeling, threading, and protein–protein docking strategies. Molecular dynamics simulations revealed that PIP2 creates a stable microdomain in a dipalmitoylphosphatidylcholine bilayer, providing TIRAP with its physiologically relevant orientation. Computed binding free energy values suggest that the affinity of PI-binding domain (PBD for PIP2 is stronger than that of TIRAP as a whole for PIP2 and that the short PI-binding motif (PBM contributes to the affinity between PBD and PIP2. Four PIP2 molecules can be accommodated by distinct lysine-rich surfaces on the dimeric PBM. Along with the known PI-binding residues (K15, K16, K31, and K32, additional positively charged residues (K34, K35, and R36 showed strong affinity toward PIP2. Lysine-to-alanine mutations at the PI-binding residues abolished TIRAP’s affinity for PIP2; however, K34, K35, and R36 consistently interacted with PIP2 headgroups through hydrogen bond (H-bond and electrostatic interactions. TIRAP exhibited a PIP2-analogous intermolecular contact and binding affinity toward PIP3, aided by an H-bond network involving K34, K35, and R36. The present study extends our understanding of TIRAP’s membrane association, which could be helpful in designing peptide decoys to block TLR2-, TLR4-, TLR7-, and TLR9-mediated autoimmune diseases.

The PH Domain of PDK1 Exhibits a Novel, Phospho-Regulated Monomer-Dimer Equilibrium With Important Implications for Kinase Domain Activation: Single Molecule and Ensemble Studies†

Science.gov (United States)

Ziemba, Brian P.; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J.

2013-01-01

Phosphoinositide-Dependent Kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4-5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric state(s) of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. The present study investigates the binding of purified WT and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single molecule and ensemble measurements. Single molecule analysis of the brightness of fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric, while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single molecule analysis of 2-D diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little protein penetration into the bilayer as observed for other PH domains. The 2-D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that enables greater protein insertion into

Specificity of Plasma Membrane Targeting by the Rous Sarcoma Virus Gag Protein

OpenAIRE

Scheifele, Lisa Z.; Rhoads, Jonathan D.; Parent, Leslie J.

2003-01-01

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affin...

Exploiting lipopolysaccharide-induced deformation of lipid bilayers to modify membrane composition and generate two-dimensional geometric membrane array patterns

Energy Technology Data Exchange (ETDEWEB)

Adams, Peter G. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Swingle, Kirstie L. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Univ. of New Mexico, Albuquerque, NM (United States); Paxton, Walter F. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Nogan, John J. [Sandia National Lab. (SNL-NM), Albuquerque, NM (United States); Stromberg, Loreen R. [Univ. of New Mexico, Albuquerque, NM (United States); Firestone, Millicent A. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); Mukundan, Harshini [Los Alamos National Lab. (LANL), Los Alamos, NM (United States); New Mexico Consortium, Los Alamos, NM (United States); Montaño, Gabriel A. [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2015-05-27

Supported lipid bilayers have proven effective as model membranes for investigating biophysical processes and in development of sensor and array technologies. The ability to modify lipid bilayers after their formation and in situ could greatly advance membrane technologies, but is difficult via current state-of-the-art technologies. Here we demonstrate a novel method that allows the controlled post-formation processing and modification of complex supported lipid bilayer arrangements, under aqueous conditions. We exploit the destabilization effect of lipopolysaccharide, an amphiphilic biomolecule, interacting with lipid bilayers to generate voids that can be backfilled to introduce desired membrane components. We further demonstrate that when used in combination with a single, traditional soft lithography process, it is possible to generate hierarchically-organized membrane domains and microscale 2-D array patterns of domains. Significantly, this technique can be used to repeatedly modify membranes allowing iterative control over membrane composition. This approach expands our toolkit for functional membrane design, with potential applications for enhanced materials templating, biosensing and investigating lipid-membrane processes.

Activation of the plasma membrane Na/H antiporter salt-overly-sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain

KAUST Repository

Quintero, Francisco J.; Martí nez-Atienza, Juliana; Villalta, Irene; Jiang, Xingyu; Kim, Woeyeon; Ali, Zhair; Fujii, Hiroaki; Mendoza, Imelda; Yun, Daejin; Zhu, Jian-Kang; Pardo, José Manuel

2011-01-01

The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2-SOS3 calcium-dependent protein kinase complex upregulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2-SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2-SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

Activation of the plasma membrane Na/H antiporter salt-overly-sensitive 1 (SOS1) by phosphorylation of an auto-inhibitory C-terminal domain

KAUST Repository

Quintero, Francisco J.

2011-01-24

The plasma membrane sodium/proton exchanger Salt-Overly-Sensitive 1 (SOS1) is a critical salt tolerance determinant in plants. The SOS2-SOS3 calcium-dependent protein kinase complex upregulates SOS1 activity, but the mechanistic details of this crucial event remain unresolved. Here we show that SOS1 is maintained in a resting state by a C-terminal auto-inhibitory domain that is the target of SOS2-SOS3. The auto-inhibitory domain interacts intramolecularly with an adjacent domain of SOS1 that is essential for activity. SOS1 is relieved from auto-inhibition upon phosphorylation of the auto-inhibitory domain by SOS2-SOS3. Mutation of the SOS2 phosphorylation and recognition site impeded the activation of SOS1 in vivo and in vitro. Additional amino acid residues critically important for SOS1 activity and regulation were identified in a genetic screen for hypermorphic alleles.

Nanoscopic morphological changes in yeast cell surfaces caused by oxidative stress: an atomic force microscopic study.

Science.gov (United States)

Canetta, Elisabetta; Walker, Graeme M; Adya, Ashok K

2009-06-01

Nanoscopic changes in the cell surface morphology of the yeasts Saccharomyces cerevisiae (strain NCYC 1681) and Schizosaccharomyces pombe (strain DVPB 1354), due to their exposure to varying concentrations of hydrogen peroxide (oxidative stress), were investigated using an atomic force microscope (AFM). Increasing hydrogen peroxide concentration led to a decrease in cell viabilities and mean cell volumes, and an increase in the surface roughness of the yeasts. In addition, AFM studies revealed that oxidative stress caused cell compression in both S. cerevisiae and Schiz. pombe cells and an increase in the number of aged yeasts. These results confirmed the importance and usefulness of AFM in investigating the morphology of stressed microbial cells at the nanoscale. The results also provided novel information on the relative oxidative stress tolerance of S. cerevisiae and Schiz. pombe.

Proteomic Analysis of Detergent Resistant Membrane Domains during Early Interaction of Macrophages with Rough and Smooth Brucella melitensis

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Lauer, Sabine A.; Iyer, Srinivas; Sanchez, Timothy; Forst, Christian V.; Bowden, Brent; Carlson, Kay; Sriranganathan, Nammalwar; Boyle, Stephen M.

2014-01-01

The plasma membrane contains discrete nanometer-sized domains that are resistant to non-ionic detergents, and which are called detergent resistant membrane domains (DRMDs) or lipid rafts. Exposure of host cells to pathogenic bacteria has been shown to induce the re-distribution of specific host proteins between DRMDs and detergent soluble membranes, which leads to the initiation of cell signaling that enable pathogens to access host cells. DRMDs have been shown to play a role in the invasion of Brucella into host macrophages and the formation of replicative phagosomes called Brucella-containing vacuoles (BCVs). In this study we sought to characterize changes to the protein expression profiles in DRMDs and to respective cellular pathways and networks of Mono Mac 6 cells in response to the adherence of rough VTRM1 and smooth 16 M B. melitensis strains. DRMDs were extracted from Mono Mac 6 cells exposed for 2 minutes at 4°C to Brucella (no infection occurs) and from unexposed control cells. Protein expression was determined using the non-gel based quantitative iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry technique. Using the identified iTRAQ proteins we performed enrichment analyses and probed constructed human biochemical networks for interactions and metabolic reactions. We identified 149 proteins, which either became enriched, depleted or whose amounts did not change in DRMDs upon Brucella exposure. Several of these proteins were distinctly enriched or depleted in DRMDs upon exposure to rough and smooth B. melitensis strains which results in the differential engagement of cellular pathways and networks immediately upon Brucella encounter. For some of the proteins such as myosin 9, small G protein signaling modulator 3, lysine-specific demethylase 5D, erlin-2, and voltage-dependent anion-selective channel protein 2, we observed extreme differential depletion or enrichment in DRMDs. The identified proteins and pathways could provide

Myosin-1A Targets to Microvilli Using Multiple Membrane Binding Motifs in the Tail Homology 1 (TH1) Domain*

Science.gov (United States)

Mazerik, Jessica N.; Tyska, Matthew J.

2012-01-01

One of the most abundant components of the enterocyte brush border is the actin-based monomeric motor, myosin-1a (Myo1a). Within brush border microvilli, Myo1a carries out a number of critical functions at the interface between membrane and actin cytoskeleton. Proper physiological function of Myo1a depends on its ability to bind to microvillar membrane, an interaction mediated by a C-terminal tail homology 1 (TH1) domain. However, little is known about the mechanistic details of the Myo1a-TH1/membrane interaction. Structure-function analysis of Myo1a-TH1 targeting in epithelial cells revealed that an N-terminal motif conserved among class I myosins and a C-terminal motif unique to Myo1a-TH1 are both required for steady state microvillar enrichment. Purified Myo1a bound to liposomes composed of phosphatidylserine and phosphoinositol 4,5-bisphosphate, with moderate affinity in a charge-dependent manner. Additionally, peptides of the N- and C-terminal regions required for targeting were able to compete with Myo1a for binding to highly charged liposomes in vitro. Single molecule total internal reflection fluorescence microscopy showed that these motifs are also necessary for slowing the membrane detachment rate in cells. Finally, Myo1a-TH1 co-localized with both lactadherin-C2 (a phosphatidylserine-binding protein) and PLCδ1-PH (a phosphoinositol 4,5-bisphosphate-binding protein) in microvilli, but only lactaderin-C2 expression reduced brush border targeting of Myo1a-TH1. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif. These data highlight the diversity of mechanisms that enable different class I myosins to target membranes in distinct biological contexts. PMID:22367206

Nanoscopic characterization of Pr2Zr2O7 at Zr sites

International Nuclear Information System (INIS)

Martinez, J.A.; Caracoche, M.C.; Rodriguez, A.M.; Rivas, P.C.; Bondioli, F.; Manfredini, T.; Ferrari, A.M.

2005-01-01

By using Perturbed Angular Correlation Spectroscopy, a suitable technique to explore internal fields at nanoscopic scale, the electric field gradients at Zr 4+ sites in the Pr 2 Zr 2 O 7 compound have been determined as a function of temperature. Three nonequivalent nanoconfigurations are present, which have been interpreted with the aid of point charge model calculations. Two of them correspond to pyrochlore - oxygen defective and perfect structures -, and the third one to the pyrochlore-related defect fluorite structure. The most abundant interaction is a disordered and fluctuating electric field gradient, which describes the oxygen defective pyrochlore. As temperature increases, its gradual and reversible transformation towards the perfect form is observed. Below 750 C the oxygen vacancies movement, which exhibits an activation energy of 0.14 eV, is assumed to be due to vacancies jumping among 48f equivalent sites. At higher temperatures the movement is interpreted as the fast diffusion of oxygen vacancies involving 48f and 8b sites, thus giving place to anionic disorder. The activation energy for this movement has been determined to be of 0.85 eV. (copyright 2005 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

Structural interactions between lipids, water and S1-S4 voltage-sensing domains.

Science.gov (United States)

Krepkiy, Dmitriy; Gawrisch, Klaus; Swartz, Kenton J

2012-11-02

Membrane proteins serve crucial signaling and transport functions, yet relatively little is known about their structures in membrane environments or how lipids interact with these proteins. For voltage-activated ion channels, X-ray structures suggest that the mobile voltage-sensing S4 helix would be exposed to the membrane, and functional studies reveal that lipid modification can profoundly alter channel activity. Here, we use solid-state NMR to investigate structural interactions of lipids and water with S1-S4 voltage-sensing domains and to explore whether lipids influence the structure of the protein. Our results demonstrate that S1-S4 domains exhibit extensive interactions with lipids and that these domains are heavily hydrated when embedded in a membrane. We also find evidence for preferential interactions of anionic lipids with S1-S4 domains and that these interactions have lifetimes on the timescale of ≤ 10(-3)s. Arg residues within S1-S4 domains are well hydrated and are positioned in close proximity to lipids, exhibiting local interactions with both lipid headgroups and acyl chains. Comparative studies with a positively charged lipid lacking a phosphodiester group reveal that this lipid modification has only modest effects on the structure and hydration of S1-S4 domains. Taken together, our results demonstrate that Arg residues in S1-S4 voltage-sensing domains reside in close proximity to the hydrophobic interior of the membrane yet are well hydrated, a requirement for carrying charge and driving protein motions in response to changes in membrane voltage. Published by Elsevier Ltd.

Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: Mapping of residues that when altered give rise to an activated enzyme

DEFF Research Database (Denmark)

Axelsen, K.B.; Venema, K.; Jah, T.

1999-01-01

in an extension of the C-terminus unique to plant H+-ATPases, Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain......The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants, The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme......+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues...

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

Science.gov (United States)

Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

1995-01-01

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

A unifying mechanism accounts for sensing of membrane curvature by BAR domains, amphipathic helices and membrane-anchored proteins

DEFF Research Database (Denmark)

Bhatia, Vikram Kjøller; Hatzakis, Nikos; Stamou, Dimitrios

2010-01-01

itself. We thus anticipate that membrane curvature will promote the redistribution of proteins that are anchored in membranes through any type of hydrophobic moiety, a thesis that broadens tremendously the implications of membrane curvature for protein sorting, trafficking and signaling in cell biology....

Estimation of adsorption-induced pore pressure and confinement in a nanoscopic slit pore by a density functional theory

Science.gov (United States)

Grégoire, David; Malheiro, Carine; Miqueu, Christelle

2018-03-01

This study aims at characterising the adsorption-induced pore pressure and confinement in nanoscopic pores by molecular non-local density functional theory (DFT). Considering its important potential industrial applications, the adsorption of methane in graphitic slit pores has been selected as the test case. While retaining the accuracy of molecular simulations at pore scale, DFT has a very low computational cost that allows obtaining highly resolved pore pressure maps as a function of both pore width and thermodynamic conditions. The dependency of pore pressure on these parameters (pore width, pressure and temperature) is carefully analysed in order to highlight the effect of each parameter on the confined fluid properties that impact the solid matrix.

Membrane fluctuations mediate lateral interaction between cadherin bonds

Science.gov (United States)

Fenz, Susanne F.; Bihr, Timo; Schmidt, Daniel; Merkel, Rudolf; Seifert, Udo; Sengupta, Kheya; Smith, Ana-Sunčana

2017-09-01

The integrity of living tissues is maintained by adhesion domains of trans-bonds formed between cadherin proteins residing on opposing membranes of neighbouring cells. These domains are stabilized by lateral cis-interactions between the cadherins on the same cell. However, the origin of cis-interactions remains perplexing since they are detected only in the context of trans-bonds. By combining experimental, analytical and computational approaches, we identify bending fluctuations of membranes as a source of long-range cis-interactions, and a regulator of trans-interactions. Specifically, nanometric membrane bending and fluctuations introduce cooperative effects that modulate the affinity and binding/unbinding rates for trans-dimerization, dramatically affecting the nucleation and growth of adhesion domains. Importantly, this regulation relies on physical principles and not on details of protein-protein interactions. These omnipresent fluctuations can thus act as a generic control mechanism in all types of cell adhesion, suggesting a hitherto unknown physiological role for recently identified active fluctuations of cellular membranes.

Constraints imposed by transmembrane domains affect enzymatic activity of membrane-associated human CD39/NTPDase1 mutants.

Science.gov (United States)

Musi, Elgilda; Islam, Naziba; Drosopoulos, Joan H F

2007-05-01

Human CD39/NTPDase1 is an endothelial cell membrane-associated nucleotidase. Its large extracellular domain rapidly metabolizes nucleotides, especially ADP released from activated platelets, inhibiting further platelet activation/recruitment. Previous studies using our recombinant soluble CD39 demonstrated the importance of residues S57, D54, and D213 for enzymatic/biological activity. We now report effects of S57A, D54A, and D213A mutations on full-length (FL)CD39 function. Enzymatic activity of alanine modified FLCD39s was less than wild-type, contrasting the enhanced activity of their soluble counterparts. Furthermore, conservative substitutions D54E and D213E led to enzymes with activities greater than the alanine modified FLCD39s, but less than wild-type. Reductions in mutant activities were primarily associated with reduced catalytic rates. Differences in enzymatic activity were not attributable to gross changes in the nucleotide binding pocket or the enzyme's ability to multimerize. Thus, composition of the active site of wild-type CD39 appears optimized for ADPase function in the context of the transmembrane domains.

A disease-causing mutation illuminates the protein membrane topology of the kidney-expressed prohibitin homology (PHB) domain protein podocin.

Science.gov (United States)

Schurek, Eva-Maria; Völker, Linus A; Tax, Judit; Lamkemeyer, Tobias; Rinschen, Markus M; Ungrue, Denise; Kratz, John E; Sirianant, Lalida; Kunzelmann, Karl; Chalfie, Martin; Schermer, Bernhard; Benzing, Thomas; Höhne, Martin

2014-04-18

Mutations in the NPHS2 gene are a major cause of steroid-resistant nephrotic syndrome, a severe human kidney disorder. The NPHS2 gene product podocin is a key component of the slit diaphragm cell junction at the kidney filtration barrier and part of a multiprotein-lipid supercomplex. A similar complex with the podocin ortholog MEC-2 is required for touch sensation in Caenorhabditis elegans. Although podocin and MEC-2 are membrane-associated proteins with a predicted hairpin-like structure and amino and carboxyl termini facing the cytoplasm, this membrane topology has not been convincingly confirmed. One particular mutation that causes kidney disease in humans (podocin(P118L)) has also been identified in C. elegans in genetic screens for touch insensitivity (MEC-2(P134S)). Here we show that both mutant proteins, in contrast to the wild-type variants, are N-glycosylated because of the fact that the mutant C termini project extracellularly. Podocin(P118L) and MEC-2(P134S) did not fractionate in detergent-resistant membrane domains. Moreover, mutant podocin failed to activate the ion channel TRPC6, which is part of the multiprotein-lipid supercomplex, indicative of the fact that cholesterol recruitment to the ion channels, an intrinsic function of both proteins, requires C termini facing the cytoplasmic leaflet of the plasma membrane. Taken together, this study demonstrates that the carboxyl terminus of podocin/MEC-2 has to be placed at the inner leaflet of the plasma membrane to mediate cholesterol binding and contribute to ion channel activity, a prerequisite for mechanosensation and the integrity of the kidney filtration barrier.

The PH domain of phosphoinositide-dependent kinase-1 exhibits a novel, phospho-regulated monomer-dimer equilibrium with important implications for kinase domain activation: single-molecule and ensemble studies.

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Ziemba, Brian P; Pilling, Carissa; Calleja, Véronique; Larijani, Banafshé; Falke, Joseph J

2013-07-16

Phosphoinositide-dependent kinase-1 (PDK1) is an essential master kinase recruited to the plasma membrane by the binding of its C-terminal PH domain to the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Membrane binding leads to PDK1 phospho-activation, but despite the central role of PDK1 in signaling and cancer biology, this activation mechanism remains poorly understood. PDK1 has been shown to exist as a dimer in cells, and one crystal structure of its isolated PH domain exhibits a putative dimer interface. It has been proposed that phosphorylation of PH domain residue T513 (or the phospho-mimetic T513E mutation) may regulate a novel PH domain dimer-monomer equilibrium, thereby converting an inactive PDK1 dimer to an active monomer. However, the oligomeric states of the PH domain on the membrane have not yet been determined, nor whether a negative charge at position 513 is sufficient to regulate its oligomeric state. This study investigates the binding of purified wild-type (WT) and T513E PDK1 PH domains to lipid bilayers containing the PIP3 target lipid, using both single-molecule and ensemble measurements. Single-molecule analysis of the brightness of the fluorescent PH domain shows that the PIP3-bound WT PH domain on membranes is predominantly dimeric while the PIP3-bound T513E PH domain is monomeric, demonstrating that negative charge at the T513 position is sufficient to dissociate the PH domain dimer and is thus likely to play a central role in PDK1 monomerization and activation. Single-molecule analysis of two-dimensional (2D) diffusion of PH domain-PIP3 complexes reveals that the dimeric WT PH domain diffuses at the same rate as a single lipid molecule, indicating that only one of its two PIP3 binding sites is occupied and there is little penetration of the protein into the bilayer as observed for other PH domains. The 2D diffusion of T513E PH domain is slower, suggesting the negative charge disrupts local structure in a way that allows

Proton Transfer in Perfluorosulfonic Acid Fuel Cell Membranes with Differing Pendant Chains and Equivalent Weights.

Science.gov (United States)

Thomaz, Joseph E; Lawler, Christian M; Fayer, Michael D

2017-05-04

Proton transfer in the nanoscopic water channels of polyelectrolyte fuel cell membranes was studied using a photoacid, 8-hydroxypyrene-1,3,6-trisulfonic acid sodium salt (HPTS), in the channels. The local environment of the probe was determined using 8-methoxypyrene-1,3,6-trisulfonic acid sodium salt (MPTS), which is not a photoacid. Three fully hydrated membranes, Nafion (DuPont) and two 3M membranes, were studied to determine the impact of different pendant chains and equivalent weights on proton transfer. Fluorescence anisotropy and excited state population decay data that characterize the local environment of the fluorescent probes and proton transfer dynamics were measured. The MPTS lifetime and anisotropy results show that most of the fluorescent probes have a bulk-like water environment with a relatively small fraction interacting with the channel wall. Measurements of the HPTS protonated and deprotonated fluorescent bands' population decays provided information on the proton transport dynamics. The decay of the protonated band from ∼0.5 ns to tens of nanoseconds is in part determined by dissociation and recombination with the HPTS, providing information on the ability of protons to move in the channels. The dissociation and recombination is manifested as a power law component in the protonated band fluorescence decay. The results show that equivalent weight differences between two 3M membranes resulted in a small difference in proton transfer. However, differences in pendant chain structure did significantly influence the proton transfer ability, with the 3M membranes displaying more facile transfer than Nafion.

BAR domain proteins regulate Rho GTPase signaling.

Science.gov (United States)

Aspenström, Pontus

2014-01-01

BAR proteins comprise a heterogeneous group of multi-domain proteins with diverse biological functions. The common denominator is the Bin-Amphiphysin-Rvs (BAR) domain that not only confers targeting to lipid bilayers, but also provides scaffolding to mold lipid membranes into concave or convex surfaces. This function of BAR proteins is an important determinant in the dynamic reconstruction of membrane vesicles, as well as of the plasma membrane. Several BAR proteins function as linkers between cytoskeletal regulation and membrane dynamics. These links are provided by direct interactions between BAR proteins and actin-nucleation-promoting factors of the Wiskott-Aldrich syndrome protein family and the Diaphanous-related formins. The Rho GTPases are key factors for orchestration of this intricate interplay. This review describes how BAR proteins regulate the activity of Rho GTPases, as well as how Rho GTPases regulate the function of BAR proteins. This mutual collaboration is a central factor in the regulation of vital cellular processes, such as cell migration, cytokinesis, intracellular transport, endocytosis, and exocytosis.

The actin homologue MreB organizes the bacterial cell membrane.

Science.gov (United States)

Strahl, Henrik; Bürmann, Frank; Hamoen, Leendert W

2014-03-07

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lipid staining techniques and spectroscopic methods, revealed that MreB filaments create specific membrane regions with increased fluidity (RIFs). Interference with these fluid lipid domains (RIFs) perturbs overall lipid homeostasis and affects membrane protein localization. The influence of MreB on membrane organization and fluidity may explain why the active movement of MreB stimulates membrane protein diffusion. These novel MreB activities add additional complexity to bacterial cell membrane organization and have implications for many membrane-associated processes.

Importance of the hexagonal lipid phase in biological membrane organization

OpenAIRE

Jouhet, Juliette

2013-01-01

Domains are present in every natural membrane. They are characterized by a distinctive protein and/or lipid composition. Their size is highly variable from the nano- to the micrometer scale. The domains confer specific properties to the membrane leading to original structure and function. The determinants leading to domain organization are therefore important but remain obscure. This review presents how the ability of lipids to organize into hexagonal II or lamellar phases can promote particu...

The Disulfide Bond Cys255-Cys279 in the Immunoglobulin-Like Domain of Anthrax Toxin Receptor 2 Is Required for Membrane Insertion of Anthrax Protective Antigen Pore.

Directory of Open Access Journals (Sweden)

Pedro Jacquez

Full Text Available Anthrax toxin receptors act as molecular clamps or switches that control anthrax toxin entry, pH-dependent pore formation, and translocation of enzymatic moieties across the endosomal membranes. We previously reported that reduction of the disulfide bonds in the immunoglobulin-like (Ig domain of the anthrax toxin receptor 2 (ANTXR2 inhibited the function of the protective antigen (PA pore. In the present study, the disulfide linkage in the Ig domain was identified as Cys255-Cys279 and Cys230-Cys315. Specific disulfide bond deletion mutants were achieved by replacing Cys residues with Ala residues. Deletion of the disulfide bond C255-C279, but not C230-C315, inhibited the PA pore-induced release of the fluorescence dyes from the liposomes, suggesting that C255-C279 is essential for PA pore function. Furthermore, we found that deletion of C255-C279 did not affect PA prepore-to-pore conversion, but inhibited PA pore membrane insertion by trapping the PA membrane-inserting loops in proteinaceous hydrophobic pockets. Fluorescence spectra of Trp59, a residue adjacent to the PA-binding motif in von Willebrand factor A (VWA domain of ANTXR2, showed that deletion of C255-C279 resulted in a significant conformational change on the receptor ectodomain. The disulfide deletion-induced conformational change on the VWA domain was further confirmed by single-particle 3D reconstruction of the negatively stained PA-receptor heptameric complexes. Together, the biochemical and structural data obtained in this study provides a mechanistic insight into the role of the receptor disulfide bond C255-C279 in anthrax toxin action. Manipulation of the redox states of the receptor, specifically targeting to C255-C279, may become a novel strategy to treat anthrax.

Size and mobility of lipid domains tuned by geometrical constraints.

Science.gov (United States)

Schütte, Ole M; Mey, Ingo; Enderlein, Jörg; Savić, Filip; Geil, Burkhard; Janshoff, Andreas; Steinem, Claudia

2017-07-25

In the plasma membrane of eukaryotic cells, proteins and lipids are organized in clusters, the latter ones often called lipid domains or "lipid rafts." Recent findings highlight the dynamic nature of such domains and the key role of membrane geometry and spatial boundaries. In this study, we used porous substrates with different pore radii to address precisely the extent of the geometric constraint, permitting us to modulate and investigate the size and mobility of lipid domains in phase-separated continuous pore-spanning membranes (PSMs). Fluorescence video microscopy revealed two types of liquid-ordered ( l o ) domains in the freestanding parts of the PSMs: ( i ) immobile domains that were attached to the pore rims and ( ii ) mobile, round-shaped l o domains within the center of the PSMs. Analysis of the diffusion of the mobile l o domains by video microscopy and particle tracking showed that the domains' mobility is slowed down by orders of magnitude compared with the unrestricted case. We attribute the reduced mobility to the geometric confinement of the PSM, because the drag force is increased substantially due to hydrodynamic effects generated by the presence of these boundaries. Our system can serve as an experimental test bed for diffusion of 2D objects in confined geometry. The impact of hydrodynamics on the mobility of enclosed lipid domains can have great implications for the formation and lateral transport of signaling platforms.

Nanoscale Membrane Domain Formation Driven by Cholesterol

Czech Academy of Sciences Publication Activity Database

Javanainen, M.; Martinez-Seara, Hector; Vattulainen, I.

2017-01-01

Roč. 7, Apr 25 (2017), č. článku 1143. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GBP208/12/G016 Institutional support: RVO:61388963 Keywords : molecular dynamics simulations * differential scanning calorimetry * pulmonary surfactant membranes Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 4.259, year: 2016 https://www.nature.com/ articles /s41598-017-01247-9

Design, construction, and characterization of high-performance membrane fusion devices with target-selectivity.

Science.gov (United States)

Kashiwada, Ayumi; Yamane, Iori; Tsuboi, Mana; Ando, Shun; Matsuda, Kiyomi

2012-01-31

Membrane fusion proteins such as the hemagglutinin glycoprotein have target recognition and fusion accelerative domains, where some synergistically working elements are essential for target-selective and highly effective native membrane fusion systems. In this work, novel membrane fusion devices bearing such domains were designed and constructed. We selected a phenylboronic acid derivative as a recognition domain for a sugar-like target and a transmembrane-peptide (Leu-Ala sequence) domain interacting with the target membrane, forming a stable hydrophobic α-helix and accelerating the fusion process. Artificial membrane fusion behavior between the synthetic devices in which pilot and target liposomes were incorporated was characterized by lipid-mixing and inner-leaflet lipid-mixing assays. Consequently, the devices bearing both the recognition and transmembrane domains brought about a remarkable increase in the initial rate for the membrane fusion compared with the devices containing the recognition domain alone. In addition, a weakly acidic pH-responsive device was also constructed by replacing three Leu residues in the transmembrane-peptide domain by Glu residues. The presence of Glu residues made the acidic pH-dependent hydrophobic α-helix formation possible as expected. The target-selective liposome-liposome fusion was accelerated in a weakly acidic pH range when the Glu-substituted device was incorporated in pilot liposomes. The use of this pH-responsive device seems to be a potential strategy for novel applications in a liposome-based delivery system. © 2011 American Chemical Society

Non-Native Metal Ion Reveals the Role of Electrostatics in Synaptotagmin 1-Membrane Interactions.

Science.gov (United States)

Katti, Sachin; Nyenhuis, Sarah B; Her, Bin; Srivastava, Atul K; Taylor, Alexander B; Hart, P John; Cafiso, David S; Igumenova, Tatyana I

2017-06-27

C2 domains are independently folded modules that often target their host proteins to anionic membranes in a Ca 2+ -dependent manner. In these cases, membrane association is triggered by Ca 2+ binding to the negatively charged loop region of the C2 domain. Here, we used a non-native metal ion, Cd 2+ , in lieu of Ca 2+ to gain insight into the contributions made by long-range Coulombic interactions and direct metal ion-lipid bridging to membrane binding. Using X-ray crystallography, NMR, Förster resonance energy transfer, and vesicle cosedimentation assays, we demonstrate that, although Cd 2+ binds to the loop region of C2A/B domains of synaptotagmin 1 with high affinity, long-range Coulombic interactions are too weak to support membrane binding of individual domains. We attribute this behavior to two factors: the stoichiometry of Cd 2+ binding to the loop regions of the C2A and C2B domains and the impaired ability of Cd 2+ to directly coordinate the lipids. In contrast, electron paramagnetic resonance experiments revealed that Cd 2+ does support membrane binding of the C2 domains in full-length synaptotagmin 1, where the high local lipid concentrations that result from membrane tethering can partially compensate for lack of a full complement of divalent metal ions and specific lipid coordination in Cd 2+ -complexed C2A/B domains. Our data suggest that long-range Coulombic interactions alone can drive the initial association of C2A/B with anionic membranes and that Ca 2+ further augments membrane binding by the formation of metal ion-lipid coordination bonds and additional Ca 2+ ion binding to the C2 domain loop regions.

Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

Science.gov (United States)

Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

2016-01-01

Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells.

Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia

Science.gov (United States)

Machta, Benjamin B.; Gray, Ellyn; Nouri, Mariam; McCarthy, Nicola L. C.; Gray, Erin M.; Miller, Ann L.; Brooks, Nicholas J.; Veatch, Sarah L.

2016-08-01

Diverse molecules induce general anesthesia with potency strongly correlated both with their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma membrane derived vesicles by lowering the critical temperature ($T_c$) for phase separation. Here we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on $T_c$. First we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described `intoxication reversers' raise $T_c$ and counter ethanol's effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that hydrostatic pressure, long known to reverse anesthesia, also raises $T_c$ in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that $\\Delta T_c$ predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.

F-BAR family proteins, emerging regulators for cell membrane dynamic changes-from structure to human diseases.

Science.gov (United States)

Liu, Suxuan; Xiong, Xinyu; Zhao, Xianxian; Yang, Xiaofeng; Wang, Hong

2015-05-09

Eukaryotic cell membrane dynamics change in curvature during physiological and pathological processes. In the past ten years, a novel protein family, Fes/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain proteins, has been identified to be the most important coordinators in membrane curvature regulation. The F-BAR domain family is a member of the Bin/Amphiphysin/Rvs (BAR) domain superfamily that is associated with dynamic changes in cell membrane. However, the molecular basis in membrane structure regulation and the biological functions of F-BAR protein are unclear. The pathophysiological role of F-BAR protein is unknown. This review summarizes the current understanding of structure and function in the BAR domain superfamily, classifies F-BAR family proteins into nine subfamilies based on domain structure, and characterizes F-BAR protein structure, domain interaction, and functional relevance. In general, F-BAR protein binds to cell membrane via F-BAR domain association with membrane phospholipids and initiates membrane curvature and scission via Src homology-3 (SH3) domain interaction with its partner proteins. This process causes membrane dynamic changes and leads to seven important cellular biological functions, which include endocytosis, phagocytosis, filopodium, lamellipodium, cytokinesis, adhesion, and podosome formation, via distinct signaling pathways determined by specific domain-binding partners. These cellular functions play important roles in many physiological and pathophysiological processes. We further summarize F-BAR protein expression and mutation changes observed in various diseases and developmental disorders. Considering the structure feature and functional implication of F-BAR proteins, we anticipate that F-BAR proteins modulate physiological and pathophysiological processes via transferring extracellular materials, regulating cell trafficking and mobility, presenting antigens, mediating extracellular matrix degradation, and transmitting

Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

International Nuclear Information System (INIS)

Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon

2006-01-01

The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K + channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments

Membrane dynamics and the regulation of epithelial cell polarity

NARCIS (Netherlands)

van der Wouden, JM; Maier, O; van IJzendoorn, SCD; Hoekstra, D

2003-01-01

Plasma membranes of epithelial cells consist of two domains, an apical and a basolateral domain, the surfaces of which differ in composition. The separation of these domains by a tight junction and the fact that specific transport pathways exist for intracellular communication between these domains

Epithelial cell-cell junctions and plasma membrane domains

NARCIS (Netherlands)

Giepmans, Ben N. G.; van Ijzendoorn, Sven C. D.

Epithelial cells form a barrier against the environment, but are also required for the regulated exchange of molecules between an organism and its surroundings. Epithelial cells are characterised by a remarkable polarization of their plasma membrane, evidenced by the appearance of structurally,

Functional interchangeability of late domains, late domain cofactors and ubiquitin in viral budding.

Directory of Open Access Journals (Sweden)

Maria Zhadina

2010-10-01

Full Text Available The membrane scission event that separates nascent enveloped virions from host cell membranes often requires the ESCRT pathway, which can be engaged through the action of peptide motifs, termed late (L- domains, in viral proteins. Viral PTAP and YPDL-like L-domains bind directly to the ESCRT-I and ALIX components of the ESCRT pathway, while PPxY motifs bind Nedd4-like, HECT-domain containing, ubiquitin ligases (e.g. WWP1. It has been unclear precisely how ubiquitin ligase recruitment ultimately leads to particle release. Here, using a lysine-free viral Gag protein derived from the prototypic foamy virus (PFV, where attachment of ubiquitin to Gag can be controlled, we show that several different HECT domains can replace the WWP1 HECT domain in chimeric ubiquitin ligases and drive budding. Moreover, artificial recruitment of isolated HECT domains to Gag is sufficient to stimulate budding. Conversely, the HECT domain becomes dispensable if the other domains of WWP1 are directly fused to an ESCRT-1 protein. In each case where budding is driven by a HECT domain, its catalytic activity is essential, but Gag ubiquitination is dispensable, suggesting that ubiquitin ligation to trans-acting proteins drives budding. Paradoxically, however, we also demonstrate that direct fusion of a ubiquitin moiety to the C-terminus of PFV Gag can also promote budding, suggesting that ubiquitination of Gag can substitute for ubiquitination of trans-acting proteins. Depletion of Tsg101 and ALIX inhibits budding that is dependent on ubiquitin that is fused to Gag, or ligated to trans-acting proteins through the action of a PPxY motif. These studies underscore the flexibility in the ways that the ESCRT pathway can be engaged, and suggest a model in which the identity of the protein to which ubiquitin is attached is not critical for subsequent recruitment of ubiquitin-binding components of the ESCRT pathway and viral budding to proceed.

The in vivo structure of biological membranes and evidence for lipid domains

Energy Technology Data Exchange (ETDEWEB)

Nickels, Jonathan D. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Chatterjee, Sneha [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Stanley, Christopher B. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Qian, Shuo [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Cheng, Xiaolin [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Myles, Dean A. A. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Standaert, Robert F. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Elkins, James G. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Katsaras, John [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States); Lopez, Daniel [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)

2017-05-23

Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium Bacillus subtilis to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron scattering spectra, we confirmed that the B. subtilis cell membrane is lamellar and determined that its average hydrophobic thickness is 24.3 ± 0.9 Ångstroms (Å). Furthermore, by creating neutron contrast within the plane of the membrane using a mixture of H- and D-fatty acids, we detected lateral features smaller than 40 nm that are consistent with the notion of lipid rafts. These experiments—performed under biologically relevant conditions—answer long-standing questions in membrane biology and illustrate a fundamentally new approach for systematic in vivo investigations of cell membrane structure.

Shallow Boomerang-shaped Influenza Hemagglutinin G13A Mutant Structure Promotes Leaky Membrane Fusion*

Science.gov (United States)

Lai, Alex L.; Tamm, Lukas K.

2010-01-01

Our previous studies showed that an angled boomerang-shaped structure of the influenza hemagglutinin (HA) fusion domain is critical for virus entry into host cells by membrane fusion. Because the acute angle of ∼105° of the wild-type fusion domain promotes efficient non-leaky membrane fusion, we asked whether different angles would still support fusion and thus facilitate virus entry. Here, we show that the G13A fusion domain mutant produces a new leaky fusion phenotype. The mutant fusion domain structure was solved by NMR spectroscopy in a lipid environment at fusion pH. The mutant adopted a boomerang structure similar to that of wild type but with a shallower kink angle of ∼150°. G13A perturbed the structure of model membranes to a lesser degree than wild type but to a greater degree than non-fusogenic fusion domain mutants. The strength of G13A binding to lipid bilayers was also intermediate between that of wild type and non-fusogenic mutants. These membrane interactions provide a clear link between structure and function of influenza fusion domains: an acute angle is required to promote clean non-leaky fusion suitable for virus entry presumably by interaction of the fusion domain with the transmembrane domain deep in the lipid bilayer. A shallower angle perturbs the bilayer of the target membrane so that it becomes leaky and unable to form a clean fusion pore. Mutants with no fixed boomerang angle interacted with bilayers weakly and did not promote any fusion or membrane perturbation. PMID:20826788

TMV nanorods with programmed longitudinal domains of differently addressable coat proteins

Science.gov (United States)

Geiger, Fania C.; Eber, Fabian J.; Eiben, Sabine; Mueller, Anna; Jeske, Holger; Spatz, Joachim P.; Wege, Christina

2013-04-01

The spacing of functional nanoscopic elements may play a fundamental role in nanotechnological and biomedical applications, but is so far rarely achieved on this scale. In this study we show that tobacco mosaic virus (TMV) and the RNA-guided self-assembly process of its coat protein (CP) can be used to establish new nanorod scaffolds that can be loaded not only with homogeneously distributed functionalities, but with distinct molecule species grouped and ordered along the longitudinal axis. The arrangement of the resulting domains and final carrier rod length both were governed by RNA-templated two-step in vitro assembly. Two selectively addressable TMV CP mutants carrying either thiol (TMVCys) or amino (TMVLys) groups on the exposed surface were engineered and shown to retain reactivity towards maleimides or NHS esters, respectively, after acetic acid-based purification and re-assembly to novel carrier rod types. Stepwise combination of CPCys and CPLys with RNA allowed fabrication of TMV-like nanorods with a controlled total length of 300 or 330 nm, respectively, consisting of adjacent longitudinal 100-to-200 nm domains of differently addressable CP species. This technology paves the way towards rod-shaped scaffolds with pre-defined, selectively reactive barcode patterns on the nanometer scale.The spacing of functional nanoscopic elements may play a fundamental role in nanotechnological and biomedical applications, but is so far rarely achieved on this scale. In this study we show that tobacco mosaic virus (TMV) and the RNA-guided self-assembly process of its coat protein (CP) can be used to establish new nanorod scaffolds that can be loaded not only with homogeneously distributed functionalities, but with distinct molecule species grouped and ordered along the longitudinal axis. The arrangement of the resulting domains and final carrier rod length both were governed by RNA-templated two-step in vitro assembly. Two selectively addressable TMV CP mutants carrying

Tetraspanins and Transmembrane Adaptor Proteins As Plasma Membrane Organizers-Mast Cell Case.

Science.gov (United States)

Halova, Ivana; Draber, Petr

2016-01-01

The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs) and transmembrane adaptor protein (TRAP)-enriched domains. Recent biophysical, microscopic, and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD)9, CD53, CD63, CD81, CD151)] or TRAPs [linker for activation of T cells (LAT), non-T cell activation linker (NTAL), and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG)] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

Multi-functional roles for the polypeptide transport associated domains of Toc75 in chloroplast protein import

Science.gov (United States)

Paila, Yamuna D; Richardson, Lynn GL; Inoue, Hitoshi; Parks, Elizabeth S; McMahon, James; Inoue, Kentaro; Schnell, Danny J

2016-01-01

Toc75 plays a central role in chloroplast biogenesis in plants as the membrane channel of the protein import translocon at the outer envelope of chloroplasts (TOC). Toc75 is a member of the Omp85 family of bacterial and organellar membrane insertases, characterized by N-terminal POTRA (polypeptide-transport associated) domains and C-terminal membrane-integrated β-barrels. We demonstrate that the Toc75 POTRA domains are essential for protein import and contribute to interactions with TOC receptors, thereby coupling preprotein recognition at the chloroplast surface with membrane translocation. The POTRA domains also interact with preproteins and mediate the recruitment of molecular chaperones in the intermembrane space to facilitate membrane transport. Our studies are consistent with the multi-functional roles of POTRA domains observed in other Omp85 family members and demonstrate that the domains of Toc75 have evolved unique properties specific to the acquisition of protein import during endosymbiotic evolution of the TOC system in plastids. DOI: http://dx.doi.org/10.7554/eLife.12631.001 PMID:26999824

Role of amphipathic helix of a herpesviral protein in membrane deformation and T cell receptor downregulation.

Directory of Open Access Journals (Sweden)

Chan-Ki Min

2008-11-01

Full Text Available Lipid rafts are membrane microdomains that function as platforms for signal transduction and membrane trafficking. Tyrosine kinase interacting protein (Tip of T lymphotropic Herpesvirus saimiri (HVS is targeted to lipid rafts in T cells and downregulates TCR and CD4 surface expression. Here, we report that the membrane-proximal amphipathic helix preceding Tip's transmembrane (TM domain mediates lipid raft localization and membrane deformation. In turn, this motif directs Tip's lysosomal trafficking and selective TCR downregulation. The amphipathic helix binds to the negatively charged lipids and induces liposome tubulation, the TM domain mediates oligomerization, and cooperation of the membrane-proximal helix with the TM domain is sufficient for localization to lipid rafts and lysosomal compartments, especially the mutivesicular bodies. These findings suggest that the membrane-proximal amphipathic helix and TM domain provide HVS Tip with the unique ability to deform the cellular membranes in lipid rafts and to downregulate TCRs potentially through MVB formation.

Molecular insights into the m-AAA protease-mediated dislocation of transmembrane helices in the mitochondrial inner membrane.

Science.gov (United States)

Lee, Seoeun; Lee, Hunsang; Yoo, Suji; Kim, Hyun

2017-12-08

Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane; thus, proper regulation of these proteins is essential for cell viability. The m -AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments, but the molecular mechanism involved in these processes remains elusive. This study investigated the role of the TM domains in the m -AAA protease by systematic replacement of one TM domain at a time in yeast. Our data indicated that replacement of the Yta10 TM2 domain abolishes membrane dislocation for only a subset of substrates, whereas replacement of the Yta12 TM2 domain impairs membrane dislocation for all tested substrates, suggesting different roles of the TM domains in each m -AAA protease subunit. Furthermore, m -AAA protease-mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate. This finding suggested that the m -AAA protease cannot dislocate large hydrophilic domains across the membrane, indicating that the membrane dislocation probably occurs in a lipid environment. In summary, this study highlights previously underappreciated biological roles of TM domains of the m -AAA proteases in mediating the recognition and dislocation of membrane-embedded substrates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

Science.gov (United States)

Panigrahi, Rashmi; Lemieux, M Joanne

2015-01-01

Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

Effects of freezing and cold acclimation on the plasma membrane of isolated protoplasts

Energy Technology Data Exchange (ETDEWEB)

Steponkus, P.L.

1991-01-01

This project focuses on lesions in the plasma membrane of protoplasts that occur during freezing to temperatures below {minus}5{degrees} which result in changes in the semipermeablity of the plasma membrane. This injury, referred to as loss of osmotic responsiveness, is associated with the formation of large, aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and lamellar-to-hexagonal{sub II} phase transitions in the plasma membrane and subtending lamellar. The goals of this project are to provide a mechanistic understanding of the mechanism by which freeze-induced dehydration effects the formation of aparticulate domains and lamellar-to-hexagonal{sub II} phase transitions and to determine the mechanisms by which cold acclimation and cryoprotectants preclude or diminish these ultrastructural changes. Our working hypothesis is the formation of aparticulate domains and lamellar-to-hexagon{sub II} phase transitions in the plasma membrane and subtending lamellae are manifestations of hydration-dependent bilayer-bilayer interactions.

Comparison of S. cerevisiae F-BAR domain structures reveals a conserved inositol phosphate binding site

Science.gov (United States)

Moravcevic, Katarina; Alvarado, Diego; Schmitz, Karl R.; Kenniston, Jon A.; Mendrola, Jeannine M.; Ferguson, Kathryn M.; Lemmon, Mark A.

2015-01-01

SUMMARY F-BAR domains control membrane interactions in endocytosis, cytokinesis, and cell signaling. Although generally thought to bind curved membranes containing negatively charged phospholipids, numerous functional studies argue that differences in lipid-binding selectivities of F-BAR domains are functionally important. Here, we compare membrane-binding properties of the S. cerevisiae F-BAR domains in vitro and in vivo. Whereas some F-BAR domains (such as Bzz1p and Hof1p F-BARs) bind equally well to all phospholipids, the F-BAR domain from the RhoGAP Rgd1p preferentially binds phosphoinositides. We determined X-ray crystal structures of F-BAR domains from Hof1p and Rgd1p, the latter bound to an inositol phosphate. The structures explain phospholipid-binding selectivity differences, and reveal an F-BAR phosphoinositide binding site that is fully conserved in a mammalian RhoGAP called Gmip, and is partly retained in certain other F-BAR domains. Our findings reveal previously unappreciated determinants of F-BAR domain lipid-binding specificity, and provide a basis for its prediction from sequence. PMID:25620000

A conserved domain in type III secretion links the cytoplasmic domain of InvA to elements of the basal body

International Nuclear Information System (INIS)

Lilic, Mirjana; Quezada, Cindy M.; Stebbins, C. Erec

2010-01-01

The cytoplasmic domain of Salmonella InvA shares homology to a recurring scaffold in the membrane-spanning components of the type II and type III secretion systems. Protein type III secretion systems (T3SSs) are organic nanosyringes that achieve an energy-dependent translocation of bacterial proteins through the two membranes of Gram-negative organisms. Examples include the pathogenic systems of animals, plants and symbiotic bacteria that inject factors into eukaryotic cells, and the flagellar export system that secretes flagellin. T3SSs possess a core of several membrane-associated proteins that are conserved across all known bacterial species that use this system. The Salmonella protein InvA is one of the most highly conserved proteins of this core of critical T3SS components. The crystal structure of a C-terminal domain of InvA reveals an unexpected homology to domains that have been repeatedly found as building blocks of other elements of the T3SS apparatus. This suggests the surprising hypothesis that evolution has produced a significant component of the apparatus structure through a series of gene-duplication and gene-rearrangement events

Tetraspanins and Transmembrane Adaptor Proteins as Plasma Membrane Organizers – Mast Cell Case

Directory of Open Access Journals (Sweden)

Ivana eHalova

2016-05-01

Full Text Available The plasma membrane contains diverse and specialized membrane domains, which include tetraspanin-enriched domains (TEMs and transmembrane adaptor protein (TRAP-enriched domains. Recent biophysical, microscopic and functional studies indicated that TEMs and TRAP-enriched domains are involved in compartmentalization of physicochemical events of such important processes as immunoreceptor signal transduction and chemotaxis. Moreover, there is evidence of a cross-talk between TEMs and TRAP-enriched domains. In this review we discuss the presence and function of such domains and their crosstalk using mast cells as a model. The combined data based on analysis of selected mast cell-expressed tetraspanins [cluster of differentiation (CD9, CD53, CD63, CD81, CD151] or TRAPs [linker for activation of T cells (LAT, non-T cell activation linker (NTAL, and phosphoprotein associated with glycosphingolipid-enriched membrane microdomains (PAG] using knockout mice or specific antibodies point to a diversity within these two families and bring evidence of the important roles of these molecules in signaling events. An example of this diversity is physical separation of two TRAPs, LAT and NTAL, which are in many aspects similar but show plasma membrane location in different microdomains in both non-activated and activated cells. Although our understanding of TEMs and TRAP-enriched domains is far from complete, pharmaceutical applications of the knowledge about these domains are under way.

Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

Science.gov (United States)

Suzuki, Kenichi G N; Ando, Hiromune; Komura, Naoko; Konishi, Miku; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Fujiwara, Takahiro K; Kusumi, Akihiro

2018-01-01

Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods. © 2018 Elsevier Inc. All rights reserved.

Effects of membrane properties on the binding activities of the HN and HC heavy-chain domains of botulinum neurotoxin A.

Science.gov (United States)

Ayyar, B Vijayalakshmi; Atassi, M Zouhair

2016-12-01

Binding behaviors of the H N and the H C domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A H N and H C regions were prepared (H N 519-845 and H C 967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of H N 519-845 to Neuro 2a cells without affecting H C 967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both H C 967-1296 and H N 519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both H C 967-1296 and H N 519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K + ) depletion buffer lowered the binding of both H C 967-1296 and H N 519-845 to the cells, but seemed to exert a more pronounced effect on the binding of H C 967-1296 than on the binding of H N 519-845. Results indicate that while both the H N and H C domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Analysis of ultrasonic techniques for the characterization of microfiltration polymeric membranes

International Nuclear Information System (INIS)

Lucas, Carla S.; Baroni, Douglas B.; Costa, Antonio M.L.M.; Bittencourt, Marcelo S.Q.

2009-01-01

The use of polymeric membranes is extremely important in several industries such as nuclear, biotechnology, chemical and pharmaceutical. In the nuclear area, for instance, systems based on membrane separation technologies are currently being used in the treatment of radioactive liquid effluent, and new technologies using membranes are being developed at a great rate. The knowledge of the physical characteristics of these membranes, such as, pore size and the pore size distribution, is very important to the membranes separation processes. Only after these characteristics are known is it possible to determine the type and to choose a particular membrane for a specific application. In this work, two ultrasonic non destructive techniques were used to determine the porosity of membranes: pulse echo and transmission. A 25 MHz immersion transducer was used. Ultrasonic signals were acquired, for both techniques, after the ultrasonic waves passed through a microfiltration polymeric membrane of pore size of 0.45 μm and thickness of 180 μm. After the emitted ultrasonic signal crossed the membrane, the received signal brought several information on the influence of the membrane porosity in the standard signal of the ultrasonic wave. The ultrasonic signals were acquired in the time domain and changed to the frequency domain by application of the Fourier Fast Transform (FFT), thus generating the material frequency spectrum. For the pulse echo technique, the ultrasonic spectrum frequency changed after the ultrasonic wave crossed the membrane. With the transmission technique there was only a displacement of the ultrasonic signal at the time domain. (author)

G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

Science.gov (United States)

Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

2017-09-01

G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular basis of the specific subcellular localization of the C2-like domain of 5-lipoxygenase.

Science.gov (United States)

Kulkarni, Shilpa; Das, Sudipto; Funk, Colin D; Murray, Diana; Cho, Wonhwa

2002-04-12

The activation of 5-lipoxygenase (5-LO) involves its calcium-dependent translocation to the nuclear envelope, where it catalyzes the two-step transformation of arachidonic acid into leukotriene A(4), leading to the synthesis of various leukotrienes. To understand the mechanism by which 5-LO is specifically targeted to the nuclear envelope, we studied the membrane binding properties of the amino-terminal domain of 5-LO, which has been proposed to have a C2 domain-like structure. The model building, electrostatic potential calculation, and in vitro membrane binding studies of the isolated C2-like domain of 5-LO and selected mutants show that this Ca(2+)-dependent domain selectively binds zwitterionic phosphatidylcholine, which is conferred by tryptophan residues (Trp(13), Trp(75), and Trp(102)) located in the putative Ca(2+)-binding loops. The spatiotemporal dynamics of the enhanced green fluorescence protein-tagged C2-like domain of 5-LO and mutants in living cells also show that the phosphatidylcholine selectivity of the C2-like domain accounts for the specific targeting of 5-LO to the nuclear envelope. Together, these results show that the C2-like domain of 5-LO is a genuine Ca(2+)-dependent membrane-targeting domain and that the subcellular localization of the domain is governed in large part by its membrane binding properties.

Lipid self-assembly and lectin-induced reorganization of the plasma membrane.

Science.gov (United States)

Sych, Taras; Mély, Yves; Römer, Winfried

2018-05-26

The plasma membrane represents an outstanding example of self-organization in biology. It plays a vital role in protecting the integrity of the cell interior and regulates meticulously the import and export of diverse substances. Its major building blocks are proteins and lipids, which self-assemble to a fluid lipid bilayer driven mainly by hydrophobic forces. Even if the plasma membrane appears-globally speaking-homogeneous at physiological temperatures, the existence of specialized nano- to micrometre-sized domains of raft-type character within cellular and synthetic membrane systems has been reported. It is hypothesized that these domains are the origin of a plethora of cellular processes, such as signalling or vesicular trafficking. This review intends to highlight the driving forces of lipid self-assembly into a bilayer membrane and the formation of small, transient domains within the plasma membrane. The mechanisms of self-assembly depend on several factors, such as the lipid composition of the membrane and the geometry of lipids. Moreover, the dynamics and organization of glycosphingolipids into nanometre-sized clusters will be discussed, also in the context of multivalent lectins, which cluster several glycosphingolipid receptor molecules and thus create an asymmetric stress between the two membrane leaflets, leading to tubular plasma membrane invaginations.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Author(s).

Structure of synaptophysin: a hexameric MARVEL-domain channel protein.

Science.gov (United States)

Arthur, Christopher P; Stowell, Michael H B

2007-06-01

Synaptophysin I (SypI) is an archetypal member of the MARVEL-domain family of integral membrane proteins and one of the first synaptic vesicle proteins to be identified and cloned. Most all MARVEL-domain proteins are involved in membrane apposition and vesicle-trafficking events, but their precise role in these processes is unclear. We have purified mammalian SypI and determined its three-dimensional (3D) structure by using electron microscopy and single-particle 3D reconstruction. The hexameric structure resembles an open basket with a large pore and tenuous interactions within the cytosolic domain. The structure suggests a model for Synaptophysin's role in fusion and recycling that is regulated by known interactions with the SNARE machinery. This 3D structure of a MARVEL-domain protein provides a structural foundation for understanding the role of these important proteins in a variety of biological processes.

Membrane topology of hedgehog acyltransferase.

Science.gov (United States)

Matevossian, Armine; Resh, Marilyn D

2015-01-23

Hedgehog acyltransferase (Hhat) is a multipass transmembrane enzyme that mediates the covalent attachment of the 16-carbon fatty acid palmitate to the N-terminal cysteine of Sonic Hedgehog (Shh). Palmitoylation of Shh by Hhat is critical for short and long range signaling. Knowledge of the topological organization of Hhat transmembrane helices would enhance our understanding of Hhat-mediated Shh palmitoylation. Bioinformatics analysis of transmembrane domains within human Hhat using 10 different algorithms resulted in highly consistent predictions in the C-terminal, but not in the N-terminal, region of Hhat. To empirically determine the topology of Hhat, we designed and exploited Hhat constructs containing either terminal or 12 different internal epitope tags. We used selective permeabilization coupled with immunofluorescence as well as a protease protection assay to demonstrate that Hhat contains 10 transmembrane domains and 2 re-entrant loops. The invariant His and highly conserved Asp residues within the membrane-bound O-acyltransferase (MBOAT) homology domain are segregated on opposite sides of the endoplasmic reticulum membrane. The localization of His-379 on the lumenal membrane surface is consistent with a role for this invariant residue in catalysis. Analysis of the activity and stability of the Hhat constructs revealed that the C-terminal MBOAT domain is especially sensitive to manipulation. Moreover, there was remarkable similarity in the overall topological organization of Hhat and ghrelin O-acyltransferase, another MBOAT family member. Knowledge of the topological organization of Hhat could serve as an important tool for further design of selective Hhat inhibitors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Subcellular membrane fluidity of Lactobacillus delbrueckii subsp. bulgaricus under cold and osmotic stress.

Science.gov (United States)

Meneghel, Julie; Passot, Stéphanie; Cenard, Stéphanie; Réfrégiers, Matthieu; Jamme, Frédéric; Fonseca, Fernanda

2017-09-01

Cryopreservation of lactic acid bacteria may lead to undesirable cell death and functionality losses. The membrane is the first target for cell injury and plays a key role in bacterial cryotolerance. This work aimed at investigating at a subcellular resolution the membrane fluidity of two populations of Lactobacillus delbrueckii subsp. bulgaricus when subjected to cold and osmotic stresses associated to freezing. Cells were cultivated at 42 °C in mild whey medium, and they were exposed to sucrose solutions of different osmolarities (300 and 1800 mOsm L -1 ) after harvest. Synchrotron fluorescence microscopy was used to measure membrane fluidity of cells labeled with the cytoplasmic membrane probe 1-[4 (trimethylamino) phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Images were acquired at 25 and 0 °C, and more than a thousand cells were individually analyzed. Results revealed that a bacterial population characterized by high membrane fluidity and a homogeneous distribution of fluidity values appeared to be positively related to freeze-thaw resistance. Furthermore, rigid domains with different anisotropy values were observed and the occurrence of these domains was more important in the freeze-sensitive bacterial population. The freeze-sensitive cells exhibited a broadening of existing highly rigid lipid domains with osmotic stress. The enlargement of domains might be ascribed to the interaction of sucrose with membrane phospholipids, leading to membrane disorganization and cell degradation.

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ.

Science.gov (United States)

Bharat, Tanmay A M; Hoffmann, Patrick C; Kukulski, Wanda

2018-04-10

Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation. Copyright © 2018 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.. All rights reserved.

The C2 domain of PKCalpha is a Ca2+ -dependent PtdIns(4,5)P2 sensing domain: a new insight into an old pathway.

Science.gov (United States)

Sánchez-Bautista, Sonia; Marín-Vicente, Consuelo; Gómez-Fernández, Juan C; Corbalán-García, Senena

2006-10-06

The C2 domain is a targeting domain that responds to intracellular Ca2+ signals in classical protein kinases (PKCs) and mediates the translocation of its host protein to membranes. Recent studies have revealed a new motif in the C2 domain, named the lysine-rich cluster, that interacts with acidic phospholipids. The purpose of this work was to characterize the molecular mechanism by which PtdIns(4,5)P2 specifically interacts with this motif. Using a combination of isothermal titration calorimetry, fluorescence resonance energy transfer and time-lapse confocal microscopy, we show here that Ca2+ specifically binds to the Ca2+ -binding region, facilitating PtdIns(4,5)P2 access to the lysine-rich cluster. The magnitude of PtdIns(4,5)P2 binding is greater than in the case of other polyphosphate phosphatidylinositols. Very importantly, the residues involved in PtdIns(4,5)P2 binding are essential for the plasma membrane localization of PKCalpha when RBL-2H3 cells are stimulated through their IgE receptors. Additionally, CFP-PH and CFP-C1 domains were used as bioprobes to demonstrate the co-existence of PtdIns(4,5)P2 and diacylglycerol in the plasma membrane, and it was shown that although a fraction of PtdIns(4,5)P2 is hydrolyzed to generate diacylglycerol and IP3, an important amount still remains in the membrane where it is available to activate PKCalpha. These findings entail revision of the currently accepted model of PKCalpha recruitment to the membrane and its activation.

Shiga toxin induces membrane reorganization and formation of long range lipid order

DEFF Research Database (Denmark)

Solovyeva, Vita; Johannes, Ludger; Simonsen, Adam Cohen

2015-01-01

membrane reordering. When Shiga toxin was added above the lipid chain melting temperature, the toxin interaction with the membrane induced rearrangement and clustering of Gb3 lipids that resulted in the long range order and alignment of lipids in gel domains. The toxin induced redistribution of Gb3 lipids...... inside gel domains is governed by the temperature at which Shiga toxin was added to the membrane: above or below the phase transition. The temperature is thus one of the critical factors controlling lipid organization and texture in the presence of Shiga toxin. Lipid chain ordering imposed by Shiga toxin...... binding can be another factor driving the reconstruction of lipid organization and crystallization of lipids inside gel domains....

EH domain of EHD1

Energy Technology Data Exchange (ETDEWEB)

Kieken, Fabien; Jovic, Marko; Naslavsky, Naava; Caplan, Steve, E-mail: scaplan@unmc.edu; Sorgen, Paul L. [University of Nebraska Medical Center, Department of Biochemistry and Molecular Biology and Eppley Cancer Center (United States)], E-mail: psorgen@unmc.edu

2007-12-15

EHD1 is a member of the mammalian C-terminal Eps15 homology domain (EH) containing protein family, and regulates the recycling of various receptors from the endocytic recycling compartment to the plasma membrane. The EH domain of EHD1 binds to proteins containing either an Asn-Pro-Phe or Asp-Pro-Phe motif, and plays an important role in the subcellular localization and function of EHD1. Thus far, the structures of five N-terminal EH domains from other proteins have been solved, but to date, the structure of the EH domains from the four C-terminal EHD family paralogs remains unknown. In this study, we have assigned the 133 C-terminal residues of EHD1, which includes the EH domain, and solved its solution structure. While the overall structure resembles that of the second of the three N-terminal Eps15 EH domains, potentially significant differences in surface charge and the structure of the tripeptide-binding pocket are discussed.

EH domain of EHD1

International Nuclear Information System (INIS)

Kieken, Fabien; Jovic, Marko; Naslavsky, Naava; Caplan, Steve; Sorgen, Paul L.

2007-01-01

EHD1 is a member of the mammalian C-terminal Eps15 homology domain (EH) containing protein family, and regulates the recycling of various receptors from the endocytic recycling compartment to the plasma membrane. The EH domain of EHD1 binds to proteins containing either an Asn-Pro-Phe or Asp-Pro-Phe motif, and plays an important role in the subcellular localization and function of EHD1. Thus far, the structures of five N-terminal EH domains from other proteins have been solved, but to date, the structure of the EH domains from the four C-terminal EHD family paralogs remains unknown. In this study, we have assigned the 133 C-terminal residues of EHD1, which includes the EH domain, and solved its solution structure. While the overall structure resembles that of the second of the three N-terminal Eps15 EH domains, potentially significant differences in surface charge and the structure of the tripeptide-binding pocket are discussed

Neutrons and model membranes

Science.gov (United States)

Fragneto, G.

2012-11-01

Current research in membrane protein biophysics highlights the emerging role of lipids in shaping membrane protein function. Cells and organisms have developed sophisticated mechanisms for controlling the lipid composition and many diseases are related to the failure of these mechanisms. One of the recent advances in the field is the discovery of the existence of coexisting micro-domains within a single membrane, important for regulating some signaling pathways. Many important properties of these domains remain poorly characterized. The characterization and analysis of bio-interfaces represent a challenge. Performing measurements on these few nanometer thick, soft, visco-elastic and dynamic systems is close to the limits of the available tools and methods. Neutron scattering techniques including small angle scattering, diffraction, reflectometry as well as inelastic methods are rapidly developing for these studies and are attracting an increasing number of biologists and biophysicists at large facilities. This manuscript will review some recent progress in the field and provide perspectives for future developments. It aims at highlighting neutron reflectometry as a versatile method to tackle questions dealing with the understanding and function of biomembranes and their components. The other important scattering methods are only briefly introduced.

Membrane interaction of retroviral Gag proteins

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Robert Alfred Dick

2014-04-01

Full Text Available Assembly of an infectious retroviral particle relies on multimerization of the Gag polyprotein at the inner leaflet of the plasma membrane. The three domains of Gag common to all retroviruses-- MA, CA, and NC-- provide the signals for membrane binding, assembly, and viral RNA packaging, respectively. These signals do not function independently of one another. For example, Gag multimerization enhances membrane binding and is more efficient when NC is interacting with RNA. MA binding to the plasma membrane is governed by several principles, including electrostatics, recognition of specific lipid head groups, hydrophobic interactions, and membrane order. HIV-1 uses many of these principles while Rous sarcoma virus (RSV appears to use fewer. This review describes the principles that govern Gag interactions with membranes, focusing on RSV and HIV-1 Gag. The review also defines lipid and membrane behavior, and discusses the complexities in determining how lipid and membrane behavior impact Gag membrane binding.

Assessing the nature of lipid raft membranes

DEFF Research Database (Denmark)

Niemelä, Perttu S; Ollila, Samuli; Hyvönen, Marja T

2007-01-01

of highly ordered lateral domains rich in sphingomyelin and cholesterol (CHOL). These domains, called functional lipid rafts, have been suggested to take part in a variety of dynamic cellular processes such as membrane trafficking, signal transduction, and regulation of the activity of membrane proteins......-scale simulations to elucidate the properties of ternary raft mixtures with CHOL, palmitoylsphingomyelin (PSM), and palmitoyloleoylphosphatidylcholine. We simulate two bilayers of 1,024 lipids for 100 ns in the liquid-ordered phase and one system of the same size in the liquid-disordered phase. The studies provide...... heterogeneity more difficult. The findings reveal aspects of the role of favored (specific) lipid-lipid interactions within rafts and clarify the prominent role of CHOL in altering the properties of the membrane locally in its neighborhood. Also, we show that the presence of PSM and CHOL in rafts leads...

The nonsteroidal anti-inflammatory drug indomethacin induces heterogeneity in lipid membranes: potential implication for its diverse biological action.

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Yong Zhou

2010-01-01

Full Text Available The nonsteroidal anti-inflammatory drug (NSAID, indomethacin (Indo, has a large number of divergent biological effects, the molecular mechanism(s for which have yet to be fully elucidated. Interestingly, Indo is highly amphiphilic and associates strongly with lipid membranes, which influence localization, structure and function of membrane-associating proteins and actively regulate cell signaling events. Thus, it is possible that Indo regulates diverse cell functions by altering micro-environments within the membrane. Here we explored the effect of Indo on the nature of the segregated domains in a mixed model membrane composed of dipalmitoyl phosphatidyl-choline (di16:0 PC, or DPPC and dioleoyl phosphatidyl-choline (di18:1 PC or DOPC and cholesterol that mimics biomembranes.Using a series of fluorescent probes in a fluorescence resonance energy transfer (FRET study, we found that Indo induced separation between gel domains and fluid domains in the mixed model membrane, possibly by enhancing the formation of gel-phase domains. This effect originated from the ability of Indo to specifically target the ordered domains in the mixed membrane. These findings were further confirmed by measuring the ability of Indo to affect the fluidity-dependent fluorescence quenching and the level of detergent resistance of membranes.Because the tested lipids are the main lipid constituents in cell membranes, the observed formation of gel phase domains induced by Indo potentially occurs in biomembranes. This marked Indo-induced change in phase behavior potentially alters membrane protein functions, which contribute to the wide variety of biological activities of Indo and other NSAIDs.

ACRATA: a novel electron transfer domain associated to apoptosis and cancer

International Nuclear Information System (INIS)

Sanchez-Pulido, Luis; Rojas, Ana M; Valencia, Alfonso; Martinez-A, Carlos; Andrade, Miguel A

2004-01-01

Recently, several members of a vertebrate protein family containing a six trans-membrane (6TM) domain and involved in apoptosis and cancer (e.g. STEAP, STAMP1, TSAP6), have been identified in Golgi and cytoplasmic membranes. The exact function of these proteins remains unknown. We related this 6TM domain to distant protein families using intermediate sequences and methods of iterative profile sequence similarity search. Here we show for the first time that this 6TM domain is homolog to the 6TM heme binding domain of both the NADPH oxidase (Nox) family and the YedZ family of bacterial oxidoreductases. This finding gives novel insights about the existence of a previously undetected electron transfer system involved in apoptosis and cancer, and suggests further steps in the experimental characterization of these evolutionarily related families

The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer.

Science.gov (United States)

Zimmermann, Kerstin; Eells, Rebecca; Heinrich, Frank; Rintoul, Stefanie; Josey, Brian; Shekhar, Prabhanshu; Lösche, Mathias; Stern, Lawrence J

2017-10-27

Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζ cyt ) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζ cyt is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζ cyt with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζ cyt to the bilayers in dynamic equilibrium: one in which ζ cyt is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζ cyt apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

Characterization of the functions and proteomes associated with membrane rafts in chicken sperm.

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Ai Ushiyama

Full Text Available Cellular membranes are heterogeneous, and this has a great impact on cellular function. Despite the central role of membrane functions in multiple cellular processes in sperm, their molecular mechanisms are poorly understood. Membrane rafts are specific membrane domains enriched in cholesterol, ganglioside GM1, and functional proteins, and they are involved in the regulation of a variety of cellular functions. Studies of the functional characterization of membrane rafts in mammalian sperm have demonstrated roles in sperm-egg binding and the acrosomal reaction. Recently, our biochemical and cell biological studies showed that membrane rafts are present and might play functional roles in chicken sperm. In this study, we isolated membrane rafts from chicken sperm as a detergent-resistant membranes (DRM floating on a density gradient in the presence of 1% Triton X-100, and characterized the function and proteomes associated with these domains. Biochemical comparison of the DRM between fresh and cryopreserved sperm demonstrated that cryopreservation induces cholesterol loss specifically from membrane rafts, indicating the functional connection with reduced post-thaw fertility in chicken sperm. Furthermore, using an avidin-biotin system, we found that sperm DRM is highly enriched in a 60 KDa single protein able to bind to the inner perivitelline layer. To identify possible roles of membrane rafts, quantitative proteomics, combined with a stable isotope dimethyl labeling approach, identified 82 proteins exclusively or relatively more associated with membrane rafts. Our results demonstrate the functional distinctions between membrane domains and provide compelling evidence that membrane rafts are involved in various cellular pathways inherent to chicken sperm.

The lipid organisation of the cell membrane

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Ladha, S.

2000-04-01

Full Text Available Lipids and proteins in biological membranes are arranged in a mosaic of domains in the membrane. These domains represent small-scale heterogeneities in composition, shape and fluidity within the plane of the membrane, over the range of hundreds of nanometers to a few micrometers. They arise from the complex interactions of the heterogeneous mixtures of phospholipids, sterols, and proteins that make up all biological membranes.Los lípidos y las proteínas en las membranas biológicas están dispuestos en un mosaico de campos en la membrana. Estos campos representan heterogeneidades a pequeña escala en la composición, forma y fluidez dentro del plano de la membrana, en un rango que va de los cientos de nanómetros a los pocos micrómetros. Estos campos se originan de las complejas interacciones de las mezclas heterogéneas de fosfolípidos, esteroles y proteínas de las que están hechas todas y cada una de las membranas biológicas.

The establishment of polarized membrane traffic in Xenopus laevis embryos.

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Roberts, S J; Leaf, D S; Moore, H P; Gerhart, J C

1992-09-01

Delineation of apical and basolateral membrane domains is a critical step in the epithelialization of the outer layer of cells in the embryo. We have examined the initiation of polarized membrane traffic in Xenopus and show that membrane traffic is not polarized in oocytes but polarized membrane domains appear at first cleavage. The following proteins encoded by injected RNA transcripts were used as markers to monitor membrane traffic: (a) VSV G, a transmembrane glycoprotein preferentially inserted into the basolateral surface of polarized epithelial cells; (b) GThy-1, a fusion protein of VSV G and Thy-1 that is localized to the apical domains of polarized epithelial cells; and (c) prolactin, a peptide hormone that is not polarly secreted. In immature oocytes, there is no polarity in the expression of VSV G or GThy-1, as shown by the constitutive expression of both proteins at the surface in the animal and vegetal hemispheres. At meiotic maturation, membrane traffic to the surface is blocked; the plasma membrane no longer accepts the vesicles synthesized by the oocyte (Leaf, D. L., S. J. Roberts, J. C. Gerhart, and H.-P. Moore. 1990. Dev. Biol. 141:1-12). When RNA transcripts are injected after fertilization, VSV G is expressed only in the internal cleavage membranes (basolateral orientation) and is excluded from the outer surface (apical orientation, original oocyte membrane). In contrast, GThy-1 and prolactin, when expressed in embryos, are inserted or released at both the outer membrane derived from the oocyte and the inner cleavage membranes. Furthermore, not all of the cleavage membrane comes from an embryonic pool of vesicles--some of the cleavage membrane comes from vesicles synthesized during oogenesis. Using prolactin as a marker, we found that a subset of vesicles synthesized during oogenesis was only released after fertilization. However, while embryonic prolactin was secreted from both apical and basolateral surfaces, the secretion of oogenic prolactin

Sphingolipid Organization in the Plasma Membrane and the Mechanisms That Influence It.

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Kraft, Mary L

2016-01-01

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with

Understanding the role of BAR and SH3 domain-containing proteins in fungi

NARCIS (Netherlands)

Gkourtsa, A.

2017-01-01

This thesis addresses the role of SH3 and BAR domain-containing proteins in different fungal species. SH3 domains are small modules that mediate protein-protein interactions and BAR domains are dimerization domains with membrane binding and bending properties. It is known that the ScRvs167 protein

Digital holographic microscopy of phase separation in multicomponent lipid membranes

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Farzam Rad, Vahideh; Moradi, Ali-Reza; Darudi, Ahmad; Tayebi, Lobat

2016-12-01

Lateral in-homogeneities in lipid compositions cause microdomains formation and change in the physical properties of biological membranes. With the presence of cholesterol and mixed species of lipids, phospholipid membranes segregate into lateral domains of liquid-ordered and liquid-disordered phases. Coupling of two-dimensional intralayer phase separations and interlayer liquid-crystalline ordering in multicomponent membranes has been previously demonstrated. By the use of digital holographic microscopy (DHMicroscopy), we quantitatively analyzed the volumetric dynamical behavior of such membranes. The specimens are lipid mixtures composed of sphingomyelin, cholesterol, and unsaturated phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine. DHMicroscopy in a transmission mode is an effective tool for quantitative visualization of phase objects. By deriving the associated phase changes, three-dimensional information on the morphology variation of lipid stacks at arbitrary time scales is obtained. Moreover, the thickness distribution of the object at demanded axial planes can be obtained by numerical focusing. Our results show that the volume evolution of lipid domains follows approximately the same universal growth law of previously reported area evolution. However, the thickness of the domains does not alter significantly by time; therefore, the volume evolution is mostly attributed to the changes in area dynamics. These results might be useful in the field of membrane-based functional materials.

Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

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Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

2018-02-20

Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

Membrane lipidome of an epithelial cell line

DEFF Research Database (Denmark)

Sampaio, Julio L; Gerl, Mathias J; Klose, Christian

2011-01-01

Tissue differentiation is an important process that involves major cellular membrane remodeling. We used Madin-Darby canine kidney cells as a model for epithelium formation and investigated the remodeling of the total cell membrane lipidome during the transition from a nonpolarized morphology...... to an epithelial morphology and vice versa. To achieve this, we developed a shotgun-based lipidomics workflow that enabled the absolute quantification of mammalian membrane lipidomes with minimal sample processing from low sample amounts. Epithelial morphogenesis was accompanied by a major shift from sphingomyelin...... to generate an apical membrane domain that serves as a protective barrier for the epithelial sheet....

High Cholesterol/Low Cholesterol: Effects in Biological Membranes: A Review.

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Subczynski, Witold K; Pasenkiewicz-Gierula, Marta; Widomska, Justyna; Mainali, Laxman; Raguz, Marija

2017-12-01

Lipid composition determines membrane properties, and cholesterol plays a major role in this determination as it regulates membrane fluidity and permeability, as well as induces the formation of coexisting phases and domains in the membrane. Biological membranes display a very diverse lipid composition, the lateral organization of which plays a crucial role in regulating a variety of membrane functions. We hypothesize that, during biological evolution, membranes with a particular cholesterol content were selected to perform certain functions in the cells of eukaryotic organisms. In this review, we discuss the major membrane properties induced by cholesterol, and their relationship to certain membrane functions.

The orientation and molecular movement of a k(+) channel voltage-sensing domain.

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Gandhi, Chris S; Clark, Eliana; Loots, Eli; Pralle, Arnd; Isacoff, Ehud Y

2003-10-30

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.

Structure and evolution of N-domains in AAA metalloproteases.

Science.gov (United States)

Scharfenberg, Franka; Serek-Heuberger, Justyna; Coles, Murray; Hartmann, Marcus D; Habeck, Michael; Martin, Jörg; Lupas, Andrei N; Alva, Vikram

2015-02-27

Metalloproteases of the AAA (ATPases associated with various cellular activities) family play a crucial role in protein quality control within the cytoplasmic membrane of bacteria and the inner membrane of eukaryotic organelles. These membrane-anchored hexameric enzymes are composed of an N-terminal domain with one or two transmembrane helices, a central AAA ATPase module, and a C-terminal Zn(2+)-dependent protease. While the latter two domains have been well studied, so far, little is known about the N-terminal regions. Here, in an extensive bioinformatic and structural analysis, we identified three major, non-homologous groups of N-domains in AAA metalloproteases. By far, the largest one is the FtsH-like group of bacteria and eukaryotic organelles. The other two groups are specific to Yme1: one found in plants, fungi, and basal metazoans and the other one found exclusively in animals. Using NMR and crystallography, we determined the subunit structure and hexameric assembly of Escherichia coli FtsH-N, exhibiting an unusual α+β fold, and the conserved part of fungal Yme1-N from Saccharomyces cerevisiae, revealing a tetratricopeptide repeat fold. Our bioinformatic analysis showed that, uniquely among these proteins, the N-domain of Yme1 from the cnidarian Hydra vulgaris contains both the tetratricopeptide repeat region seen in basal metazoans and a region of homology to the N-domains of animals. Thus, it is a modern-day representative of an intermediate in the evolution of animal Yme1 from basal eukaryotic precursors. Copyright © 2015. Published by Elsevier Ltd.

Localization of the fourth membrane spanning domain as a ligand binding site in the human platelet α2-adrenergic receptor

International Nuclear Information System (INIS)

Matsui, Hiroaki; Lefkowitz, R.J.; Caron, M.G.; Regan, J.W.

1989-01-01

The human platelet α 2 -adrenergic receptor is an integral membrane protein which binds epinephrine. The gene for this receptor has been cloned, and the primary structure is thus known. A model of its secondary structure predicts that the receptor has seven transmembrane spanning domains. By covalent labeling and peptide mapping, the authors have identified a region of the receptor that is directly involved with ligand binding. Partially purified preparations of the receptor were covalently radiolabeled with either of two specific photoaffinity ligands: [ 3 H]SKF 102229 (an antagonist) or p-azido[ 3 H]clonidine (an agonist). The radiolabeled receptors were then digested with specific endopeptidases, and peptides containing the covalently bound radioligands were identified. Lysylendopeptidase treatment of [ 3 H]SKF 102229 labeled receptor yielded one peptide of M r 2400 as the product of a complete digest. Endopeptidase Arg-C gave a labeled peptide of M r 4000, which was further digested to the M r 2400 peptide by additional treatment with lysylendopeptidase. Using p-azido[ 3 H]clonidine-labeled receptor, a similar M r 2400 peptide was obtained by lysylendopeptidase cleavage. This M r 2400 peptide corresponds to the fourth transmembrane spanning domain of the receptor. These data suggest that this region forms part of the ligand binding domain of the human platelet α 2 -adrenergic receptor

ACRATA: a novel electron transfer domain associated to apoptosis and cancer

Directory of Open Access Journals (Sweden)

Martinez-A Carlos

2004-12-01

Full Text Available Abstract Background Recently, several members of a vertebrate protein family containing a six trans-membrane (6TM domain and involved in apoptosis and cancer (e.g. STEAP, STAMP1, TSAP6, have been identified in Golgi and cytoplasmic membranes. The exact function of these proteins remains unknown. Methods We related this 6TM domain to distant protein families using intermediate sequences and methods of iterative profile sequence similarity search. Results Here we show for the first time that this 6TM domain is homolog to the 6TM heme binding domain of both the NADPH oxidase (Nox family and the YedZ family of bacterial oxidoreductases. Conclusions This finding gives novel insights about the existence of a previously undetected electron transfer system involved in apoptosis and cancer, and suggests further steps in the experimental characterization of these evolutionarily related families.

Hydrogen-deuterium exchange and mass spectrometry reveal the pH-dependent conformational changes of diphtheria toxin T domain.

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Li, Jing; Rodnin, Mykola V; Ladokhin, Alexey S; Gross, Michael L

2014-11-04

The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our "standard condition" (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W(+)-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8-9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8-9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain.

Hydrogen–Deuterium Exchange and Mass Spectrometry Reveal the pH-Dependent Conformational Changes of Diphtheria Toxin T Domain

Science.gov (United States)

2015-01-01

The translocation (T) domain of diphtheria toxin plays a critical role in moving the catalytic domain across the endosomal membrane. Translocation/insertion is triggered by a decrease in pH in the endosome where conformational changes of T domain occur through several kinetic intermediates to yield a final trans-membrane form. High-resolution structural studies are only applicable to the static T-domain structure at physiological pH, and studies of the T-domain translocation pathway are hindered by the simultaneous presence of multiple conformations. Here, we report the application of hydrogen–deuterium exchange mass spectrometry (HDX-MS) for the study of the pH-dependent conformational changes of the T domain in solution. Effects of pH on intrinsic HDX rates were deconvolved by converting the on-exchange times at low pH into times under our “standard condition” (pH 7.5). pH-Dependent HDX kinetic analysis of T domain clearly reveals the conformational transition from the native state (W-state) to a membrane-competent state (W+-state). The initial transition occurs at pH 6 and includes the destabilization of N-terminal helices accompanied by the separation between N- and C-terminal segments. The structural rearrangements accompanying the formation of the membrane-competent state expose a hydrophobic hairpin (TH8–9) to solvent, prepare it to insert into the membrane. At pH 5.5, the transition is complete, and the protein further unfolds, resulting in the exposure of its C-terminal hydrophobic TH8–9, leading to subsequent aggregation in the absence of membranes. This solution-based study complements high resolution crystal structures and provides a detailed understanding of the pH-dependent structural rearrangement and acid-induced oligomerization of T domain. PMID:25290210

Protein-lipid interactions: from membrane domains to cellular networks

National Research Council Canada - National Science Library

Tamm, Lukas K

2005-01-01

... membranes is the lipid bilayer. Embedded in the fluid lipid bilayer are proteins of various shapes and traits. This volume illuminates from physical, chemical and biological angles the numerous - mostly quite weak - interactions between lipids, proteins, and proteins and lipids that define the delicate, highly dynamic and yet so stable fabri...

Long-chain GM1 gangliosides alter transmembrane domain registration through interdigitation

Czech Academy of Sciences Publication Activity Database

Manna, M.; Javanainen, M.; Martinez-Seara Monne, Hector; Gabius, H. J.; Rog, T.; Vattulainen, I.

2017-01-01

Roč. 1859, č. 5 (2017), s. 870-878 ISSN 0005-2736 Institutional support: RVO:61388963 Keywords : glycosphingolipid * cholesterol * membrane domain * membrane registry * molecular dynamics * computer simulations Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 3.498, year: 2016

Sequence diversity and natural selection at domain I of the apical membrane antigen 1 among Indian Plasmodium falciparum populations

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Kumar Ashwani

2007-11-01

Full Text Available Abstract Background The Plasmodium falciparum apical membrane antigen 1 (AMA1 is a leading malaria vaccine candidate antigen. The complete AMA1 protein is comprised of three domains where domain I exhibits high sequence polymorphism and is thus named as the hyper-variable region (HVR. The present study describes the extent of genetic polymorphism and natural selection at domain I of the ama1 gene among Indian P. falciparum isolates. Methods The part of the ama1 gene covering domain I was PCR amplified and sequenced from 157 P. falciparum isolates collected from five different geographical regions of India. Statistical and phylogenetic analyses of the sequences were done using DnaSP ver. 4. 10. 9 and MEGA version 3.0 packages. Results A total of 57 AMA1 haplotypes were observed among 157 isolates sequenced. Forty-six of these 57 haplotypes are being reported here for the first time. The parasites collected from the high malaria transmission areas (Assam, Orissa, and Andaman and Nicobar Islands showed more haplotypes (H and nucleotide diversity π as compared to low malaria transmission areas (Uttar Pradesh and Goa. The comparison of all five Indian P. falciparum subpopulations indicated moderate level of genetic differentiation and limited gene flow (Fixation index ranging from 0.048 to 0.13 between populations. The difference between rates of non-synonymous and synonymous mutations, Tajima's D and McDonald-Kreitman test statistics suggested that the diversity at domain I of the AMA1 antigen is due to positive natural selection. The minimum recombination events were also high indicating the possible role of recombination in generating AMA1 allelic diversity. Conclusion The level of genetic diversity and diversifying selection were higher in Assam, Orissa, and Andaman and Nicobar Islands populations as compared to Uttar Pradesh and Goa. The amounts of gene flow among these populations were moderate. The data reported here will be valuable for the

Permeability and Selectivity of PPO/Graphene Composites as Mixed Matrix Membranes for CO2 Capture and Gas Separation

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Riccardo Rea

2018-01-01

Full Text Available We fabricated novel composite (mixed matrix membranes based on a permeable glassy polymer, Poly(2,6-dimethyl-1,4-phenylene oxide (PPO, and variable loadings of few-layer graphene, to test their potential in gas separation and CO2 capture applications. The permeability, selectivity and diffusivity of different gases as a function of graphene loading, from 0.3 to 15 wt %, was measured at 35 and 65 °C. Samples with small loadings of graphene show a higher permeability and He/CO2 selectivity than pure PPO, due to a favorable effect of the nanofillers on the polymer morphology. Higher amounts of graphene lower the permeability of the polymer, due to the prevailing effect of increased tortuosity of the gas molecules in the membrane. Graphene also allows dramatically reducing the increase of permeability with temperature, acting as a “stabilizer” for the polymer matrix. Such effect reduces the temperature-induced loss of size-selectivity for He/N2 and CO2/N2, and enhances the temperature-induced increase of selectivity for He/CO2. The study confirms that, as observed in the case of other graphene-based mixed matrix glassy membranes, the optimal concentration of graphene in the polymer is below 1 wt %. Below such threshold, the morphology of the nanoscopic filler added in solution affects positively the glassy chains packing, enhancing permeability and selectivity, and improving the selectivity of the membrane at increasing temperatures. These results suggest that small additions of graphene to polymers can enhance their permselectivity and stabilize their properties.

Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature

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Philip P. Cheney

2017-03-01

Full Text Available The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE and hexadecanoic acid (HDA, using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature. Both lipids studied contain 16 carbon, saturated tails and a head group tag for fluorescence microscopy measurements. The accumulation of lipids at curvatures ranging from 28 nm to 55 nm radii was measured and fluorescein labeled DHPE accumulated more than fluorescein labeled HDA at regions of membrane curvature. We then tested whether single biotinylated DHPE molecules sense curvature using single particle tracking methods. Similar to groups of fluorescein labeled DHPE accumulating at curvature, the dynamics of single molecules of biotinylated DHPE was also affected by membrane curvature and highly confined motion was observed.

Extended synaptotagmins are Ca2+-dependent lipid transfer proteins at membrane contact sites.

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Yu, Haijia; Liu, Yinghui; Gulbranson, Daniel R; Paine, Alex; Rathore, Shailendra S; Shen, Jingshi

2016-04-19

Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca(2+), allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca(2+) In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum-plasma membrane contact sites, are Ca(2+)-dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca(2+) E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca(2+)-bound C2 domains. Strikingly, the Ca(2+)-regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca(2+)sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca(2+)regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca(2+)-dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca(2+)regulation.

Randomly organized lipids and marginally stable proteins: a coupling of weak interactions to optimize membrane signaling.

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Rice, Anne M; Mahling, Ryan; Fealey, Michael E; Rannikko, Anika; Dunleavy, Katie; Hendrickson, Troy; Lohese, K Jean; Kruggel, Spencer; Heiling, Hillary; Harren, Daniel; Sutton, R Bryan; Pastor, John; Hinderliter, Anne

2014-09-01

Eukaryotic lipids in a bilayer are dominated by weak cooperative interactions. These interactions impart highly dynamic and pliable properties to the membrane. C2 domain-containing proteins in the membrane also interact weakly and cooperatively giving rise to a high degree of conformational plasticity. We propose that this feature of weak energetics and plasticity shared by lipids and C2 domain-containing proteins enhance a cell's ability to transduce information across the membrane. We explored this hypothesis using information theory to assess the information storage capacity of model and mast cell membranes, as well as differential scanning calorimetry, carboxyfluorescein release assays, and tryptophan fluorescence to assess protein and membrane stability. The distribution of lipids in mast cell membranes encoded 5.6-5.8bits of information. More information resided in the acyl chains than the head groups and in the inner leaflet of the plasma membrane than the outer leaflet. When the lipid composition and information content of model membranes were varied, the associated C2 domains underwent large changes in stability and denaturation profile. The C2 domain-containing proteins are therefore acutely sensitive to the composition and information content of their associated lipids. Together, these findings suggest that the maximum flow of signaling information through the membrane and into the cell is optimized by the cooperation of near-random distributions of membrane lipids and proteins. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova. Copyright © 2014 Elsevier B.V. All rights reserved.

Mechanisms of Membrane Binding of Small GTPase K-Ras4B Farnesylated Hypervariable Region*

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Jang, Hyunbum; Abraham, Sherwin J.; Chavan, Tanmay S.; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I.; Nussinov, Ruth; Gaponenko, Vadim

2015-01-01

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. PMID:25713064

Plant lipid environment and membrane enzymes: the case of the plasma membrane H+-ATPase.

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Morales-Cedillo, Francisco; González-Solís, Ariadna; Gutiérrez-Angoa, Lizbeth; Cano-Ramírez, Dora Luz; Gavilanes-Ruiz, Marina

2015-04-01

Several lipid classes constitute the universal matrix of the biological membranes. With their amphipathic nature, lipids not only build the continuous barrier that confers identity to every cell and organelle, but they are also active actors that modulate the activity of the proteins immersed in the lipid bilayer. The plasma membrane H(+)-ATPase, an enzyme from plant cells, is an excellent example of a transmembrane protein whose activity is influenced by the hydrophilic compartments at both sides of the membrane and by the hydrophobic domains of the lipid bilayer. As a result, an extensive documentation of the effect of numerous amphiphiles in the enzyme activity can be found. Detergents, membrane glycerolipids, and sterols can produce activation or inhibition of the enzyme activity. In some cases, these effects are associated with the lipids of the membrane bulk, but in others, a direct interaction of the lipid with the protein is involved. This review gives an account of reports related to the action of the membrane lipids on the H(+)-ATPase activity.

Comparing autotransporter β-domain configurations for their capacity to secrete heterologous proteins to the cell surface.

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Wouter S P Jong

Full Text Available Monomeric autotransporters have been extensively used for export of recombinant proteins to the cell surface of Gram-negative bacteria. A bottleneck in the biosynthesis of such constructs is the passage of the outer membrane, which is facilitated by the β-domain at the C terminus of an autotransporter in conjunction with the Bam complex in the outer membrane. We have evaluated eight β-domain constructs for their capacity to secrete fused proteins to the cell surface. These constructs derive from the monomeric autotransporters Hbp, IgA protease, Ag43 and EstA and the trimeric autotransporter Hia, which all were selected because they have been previously used for secretion of recombinant proteins. We fused three different protein domains to the eight β-domain constructs, being a Myc-tag, the Hbp passenger and a nanobody or VHH domain, and assessed expression, membrane insertion and surface exposure. Our results show that expression levels differed considerably between the constructs tested. The constructs that included the β-domains of Hbp and IgA protease appeared the most efficient and resulted in expression levels that were detectable on Coomassie-stained SDS-PAGE gels. The VHH domain appeared the most difficult fusion partner to export, probably due to its complex immunoglobulin-like structure with a tertiary structure stabilized by an intramolecular disulfide bond. Overall, the Hbp β-domain compared favorably in exporting the fused recombinant proteins, because it showed in every instance tested a good level of expression, stable membrane insertion and clear surface exposure.

Evolution and development of model membranes for physicochemical and functional studies of the membrane lateral heterogeneity.

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Morigaki, Kenichi; Tanimoto, Yasushi

2018-03-14

One of the main questions in the membrane biology is the functional roles of membrane heterogeneity and molecular localization. Although segregation and local enrichment of protein/lipid components (rafts) have been extensively studied, the presence and functions of such membrane domains still remain elusive. Along with biochemical, cell observation, and simulation studies, model membranes are emerging as an important tool for understanding the biological membrane, providing quantitative information on the physicochemical properties of membrane proteins and lipids. Segregation of fluid lipid bilayer into liquid-ordered (Lo) and liquid-disordered (Ld) phases has been studied as a simplified model of raft in model membranes, including giant unilamellar vesicles (GUVs), giant plasma membrane vesicles (GPMVs), and supported lipid bilayers (SLB). Partition coefficients of membrane proteins between Lo and Ld phases were measured to gauze their affinities to lipid rafts (raftophilicity). One important development in model membrane is patterned SLB based on the microfabrication technology. Patterned Lo/Ld phases have been applied to study the partition and function of membrane-bound molecules. Quantitative information of individual molecular species attained by model membranes is critical for elucidating the molecular functions in the complex web of molecular interactions. The present review gives a short account of the model membranes developed for studying the lateral heterogeneity, especially focusing on patterned model membranes on solid substrates. Copyright © 2018 Elsevier B.V. All rights reserved.

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

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

2015-08-18

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

Protein-membrane interaction and fatty acid transfer from intestinal fatty acid-binding protein to membranes. Support for a multistep process.

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Falomir-Lockhart, Lisandro J; Laborde, Lisandro; Kahn, Peter C; Storch, Judith; Córsico, Betina

2006-05-19

Fatty acid transfer from intestinal fatty acid-binding protein (IFABP) to phospholipid membranes occurs during protein-membrane collisions. Electrostatic interactions involving the alpha-helical "portal" region of the protein have been shown to be of great importance. In the present study, the role of specific lysine residues in the alpha-helical region of IFABP was directly examined. A series of point mutants in rat IFABP was engineered in which the lysine positive charges in this domain were eliminated or reversed. Using a fluorescence resonance energy transfer assay, we analyzed the rates and mechanism of fatty acid transfer from wild type and mutant proteins to acceptor membranes. Most of the alpha-helical domain mutants showed slower absolute fatty acid transfer rates to zwitterionic membranes, with substitution of one of the lysines of the alpha2 helix, Lys27, resulting in a particularly dramatic decrease in the fatty acid transfer rate. Sensitivity to negatively charged phospholipid membranes was also reduced, with charge reversal mutants in the alpha2 helix the most affected. The results support the hypothesis that the portal region undergoes a conformational change during protein-membrane interaction, which leads to release of the bound fatty acid to the membrane and that the alpha2 segment is of particular importance in the establishment of charge-charge interactions between IFABP and membranes. Cross-linking experiments with a phospholipid-photoactivable reagent underscored the importance of charge-charge interactions, showing that the physical interaction between wild-type intestinal fatty acid-binding protein and phospholipid membranes is enhanced by electrostatic interactions. Protein-membrane interactions were also found to be enhanced by the presence of ligand, suggesting different collisional complex structures for holo- and apo-IFABP.

The Arf-GDP-regulated recruitment of GBF1 to Golgi membranes requires domains HDS1 and HDS2 and a Golgi-localized protein receptor.

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Quilty, Douglas; Chan, Calvin J; Yurkiw, Katherine; Bain, Alexandra; Babolmorad, Ghazal; Melançon, Paul

2018-04-19

We previously proposed a novel mechanism by which the enzyme Golgi-specific Brefeldin A resistance factor 1 (GBF1) is recruited to the membranes of the cis -Golgi, based on in vivo experiments. Here, we extended our in vivo analysis on the production of regulatory Arf-GDP and observed that ArfGAP2 and ArfGAP3 do not play a role in GBF1 recruitment. We confirm that Arf-GDP localization is critical, as a TGN-localized Arf-GDP mutant protein fails to promote GBF1 recruitment. We also reported the establishment of an in vitro GBF1 recruitment assay that supports the regulation of GBF1 recruitment by Arf-GDP. This in vitro assay yielded further evidence for the requirement of a Golgi-localized protein because heat denaturation or protease treatment of Golgi membranes abrogated GBF1 recruitment. Finally, combined in vivo and in vitro measurements indicated that the recruitment to Golgi membranes via a putative receptor requires only the HDS1 and HDS2 domains in the C-terminal half of GBF1. © 2018. Published by The Company of Biologists Ltd.

The actin homologue MreB organizes the bacterial cell membrane

NARCIS (Netherlands)

Strahl, H.; Burmann, F.; Hamoen, L.W.

2014-01-01

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate

Nanodefects of membranes cause destruction of packed red blood cells during long-term storage

International Nuclear Information System (INIS)

Kozlova, Elena; Chernysh, Aleksandr; Moroz, Victor; Sergunova, Victoria; Gudkova, Olga; Kuzovlev, Artem

2015-01-01

Packed red blood cells (PRBC) are used for blood transfusion. PRBC were stored for 30 days under 4 °C in hermetic blood bags with CPD anticoagulant-preservative solution. Hematocrit was 50–55%. The distortions of PRBC membranes nanostructure and cells morphology during storage were studied by atomic force microscopy. Basic measurements were performed at the day 2, 6, 9, 16, 23 and 30 of storage and additionally 2–3 days after it. Topological defects occurred on RBC membranes by day 9. They appeared as domains with grain-like structures (“grains”) sized up to 200 nm. These domains were appeared in almost all cells. Later these domains merged and formed large defects on cells. It was the formation of domains with the “grains” which was onset process leading eventually to destruction of PRBC. Possible mechanisms of transformation of PRBC and their membrane are related to the alterations of spectrin cytoskeleton. During this storage period potassium ions and lactat concentrations increased, pH decreased, intracellular concentration of reduced glutathione diminished in the preservative solution. Changes of PRBC morphology were detected within the entire period of PRBC storage. Discocytes predominated at the days 1 and 2. By day 30 PRBC transformed into irreversible echinocytes and spheroechinocytes. Study of defects of membranes nanostructure may form the basis of assessing the quality of the stored PRBC. This method may allow to work out the best recommendations for blood transfusion. - Highlights: • Domains with “grains” are formed on membranes surface on 9–16 days of PRBC storage. • The development of domains is the reason of irreversible changes of PRBC structure. • The origin of domains is the consequence of alterations of spectrin cytoskeleton. • Study of nanostructure may form basis of assessing the quality of the stored PRBC

Nanodefects of membranes cause destruction of packed red blood cells during long-term storage

Energy Technology Data Exchange (ETDEWEB)

Kozlova, Elena, E-mail: waterlake@mail.ru [V.A. Negovsky Scientific Research Institute of General Reanimatology, Moscow (Russian Federation); I.M. Sechenov First Moscow State Medical University, Moscow (Russian Federation); Chernysh, Aleksandr [V.A. Negovsky Scientific Research Institute of General Reanimatology, Moscow (Russian Federation); I.M. Sechenov First Moscow State Medical University, Moscow (Russian Federation); Moroz, Victor; Sergunova, Victoria; Gudkova, Olga; Kuzovlev, Artem [V.A. Negovsky Scientific Research Institute of General Reanimatology, Moscow (Russian Federation)

2015-10-01

Packed red blood cells (PRBC) are used for blood transfusion. PRBC were stored for 30 days under 4 °C in hermetic blood bags with CPD anticoagulant-preservative solution. Hematocrit was 50–55%. The distortions of PRBC membranes nanostructure and cells morphology during storage were studied by atomic force microscopy. Basic measurements were performed at the day 2, 6, 9, 16, 23 and 30 of storage and additionally 2–3 days after it. Topological defects occurred on RBC membranes by day 9. They appeared as domains with grain-like structures (“grains”) sized up to 200 nm. These domains were appeared in almost all cells. Later these domains merged and formed large defects on cells. It was the formation of domains with the “grains” which was onset process leading eventually to destruction of PRBC. Possible mechanisms of transformation of PRBC and their membrane are related to the alterations of spectrin cytoskeleton. During this storage period potassium ions and lactat concentrations increased, pH decreased, intracellular concentration of reduced glutathione diminished in the preservative solution. Changes of PRBC morphology were detected within the entire period of PRBC storage. Discocytes predominated at the days 1 and 2. By day 30 PRBC transformed into irreversible echinocytes and spheroechinocytes. Study of defects of membranes nanostructure may form the basis of assessing the quality of the stored PRBC. This method may allow to work out the best recommendations for blood transfusion. - Highlights: • Domains with “grains” are formed on membranes surface on 9–16 days of PRBC storage. • The development of domains is the reason of irreversible changes of PRBC structure. • The origin of domains is the consequence of alterations of spectrin cytoskeleton. • Study of nanostructure may form basis of assessing the quality of the stored PRBC.

Electroporation-Induced Cell Modifications Detected with THz Time-Domain Spectroscopy

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Romeo, Stefania; Vernier, P. Thomas; Zeni, Olga

2018-04-01

Electroporation (electropermeabilization) increases the electrical conductivity of biological cell membranes and lowers transport barriers for normally impermeant materials. Molecular simulations suggest that electroporation begins with the reorganization of water and lipid head group dipoles in the phospholipid bilayer interface, driven by an externally applied electric field, and the evolution of the resulting defects into water-filled, lipid pores. The interior of the electroporated membrane thus contains water, which should provide a signature for detection of the electropermeabilized state. In this feasibility study, we use THz time-domain spectroscopy, a powerful tool for investigating biomolecular systems and their interactions with water, to detect electroporation in human cells subjected to permeabilizing pulsed electric fields (PEFs). The time-domain response of electroporated human monocytes was acquired with a commercial THz, time-domain spectrometer. For each sample, frequency spectra were calculated, and the absorption coefficient and refractive index were extracted in the frequency range between 0.2 and 1.5 THz. This analysis reveals a higher absorption of THz radiation by PEF-exposed cells, with respect to sham-exposed ones, consistent with the intrusion of water into the cell through the permeabilized membrane that is presumed to be associated with electroporation.

Membrane accessibility of glutathione

DEFF Research Database (Denmark)

Garcia, Almudena; Eljack, N., D.; Sani, ND

2015-01-01

Regulation of the ion pumping activity of the Na(+),K(+)-ATPase is crucial to the survival of animal cells. Recent evidence has suggested that the activity of the enzyme could be controlled by glutathionylation of cysteine residue 45 of the β-subunit. Crystal structures so far available indicate...... that this cysteine is in a transmembrane domain of the protein. Here we have analysed via fluorescence and NMR spectroscopy as well as molecular dynamics simulations whether glutathione is able to penetrate into the interior of a lipid membrane. No evidence for any penetration of glutathione into the membrane...

Transmembrane helices can induce domain formation in crowded model membranes

NARCIS (Netherlands)

Domanski, Jan; Marrink, Siewert J.; Schäfer, Lars V.

We studied compositionally heterogeneous multi-component model membranes comprised of saturated lipids, unsaturated lipids, cholesterol, and a-helical TM protein models using coarse-grained molecular dynamics simulations. Reducing the mismatch between the length of the saturated and unsaturated

Probing Growth-Induced Anisotropic Thermal Transport in High-Quality CVD Diamond Membranes by Multifrequency and Multiple-Spot-Size Time-Domain Thermoreflectance.

Science.gov (United States)

Cheng, Zhe; Bougher, Thomas; Bai, Tingyu; Wang, Steven Y; Li, Chao; Yates, Luke; Foley, Brian M; Goorsky, Mark; Cola, Baratunde A; Faili, Firooz; Graham, Samuel

2018-02-07

The maximum output power of GaN-based high-electron mobility transistors is limited by high channel temperature induced by localized self-heating, which degrades device performance and reliability. Chemical vapor deposition (CVD) diamond is an attractive candidate to aid in the extraction of this heat and in minimizing the peak operating temperatures of high-power electronics. Owing to its inhomogeneous structure, the thermal conductivity of CVD diamond varies along the growth direction and can differ between the in-plane and out-of-plane directions, resulting in a complex three-dimensional (3D) distribution. Depending on the thickness of the diamond and size of the electronic device, this 3D distribution may impact the effectiveness of CVD diamond in device thermal management. In this work, time-domain thermoreflectance is used to measure the anisotropic thermal conductivity of an 11.8 μm-thick high-quality CVD diamond membrane from its nucleation side. Starting with a spot-size diameter larger than the thickness of the membrane, measurements are made at various modulation frequencies from 1.2 to 11.6 MHz to tune the heat penetration depth and sample the variation in thermal conductivity. We then analyze the data by creating a model with the membrane divided into ten sublayers and assume isotropic thermal conductivity in each sublayer. From this, we observe a two-dimensional gradient of the depth-dependent thermal conductivity for this membrane. The local thermal conductivity goes beyond 1000 W/(m K) when the distance from the nucleation interface only reaches 3 μm. Additionally, by measuring the same region with a smaller spot size at multiple frequencies, the in-plane and cross-plane thermal conductivities are extracted. Through this use of multiple spot sizes and modulation frequencies, the 3D anisotropic thermal conductivity of CVD diamond membrane is experimentally obtained by fitting the experimental data to a thermal model. This work provides an improved

SH2 Domains Serve as Lipid-Binding Modules for pTyr-Signaling Proteins.

Science.gov (United States)

Park, Mi-Jeong; Sheng, Ren; Silkov, Antonina; Jung, Da-Jung; Wang, Zhi-Gang; Xin, Yao; Kim, Hyunjin; Thiagarajan-Rosenkranz, Pallavi; Song, Seohyeon; Yoon, Youngdae; Nam, Wonhee; Kim, Ilshin; Kim, Eui; Lee, Dong-Gyu; Chen, Yong; Singaram, Indira; Wang, Li; Jang, Myoung Ho; Hwang, Cheol-Sang; Honig, Barry; Ryu, Sungho; Lorieau, Justin; Kim, You-Me; Cho, Wonhwa

2016-04-07

The Src-homology 2 (SH2) domain is a protein interaction domain that directs myriad phosphotyrosine (pY)-signaling pathways. Genome-wide screening of human SH2 domains reveals that ∼90% of SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity. They bind lipids using surface cationic patches separate from pY-binding pockets, thus binding lipids and the pY motif independently. The patches form grooves for specific lipid headgroup recognition or flat surfaces for non-specific membrane binding and both types of interaction are important for cellular function and regulation of SH2 domain-containing proteins. Cellular studies with ZAP70 showed that multiple lipids bind its C-terminal SH2 domain in a spatiotemporally specific manner and thereby exert exquisite spatiotemporal control over its protein binding and signaling activities in T cells. Collectively, this study reveals how lipids control SH2 domain-mediated cellular protein-protein interaction networks and suggest a new strategy for therapeutic modulation of pY-signaling pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

Microdomains in the membrane landscape shape antigen-presenting cell function.

Science.gov (United States)

Zuidscherwoude, Malou; de Winde, Charlotte M; Cambi, Alessandra; van Spriel, Annemiek B

2014-02-01

The plasma membrane of immune cells is a highly organized cell structure that is key to the initiation and regulation of innate and adaptive immune responses. It is well-established that immunoreceptors embedded in the plasma membrane have a nonrandom spatial distribution that is important for coupling to components of intracellular signaling cascades. In the last two decades, specialized membrane microdomains, including lipid rafts and TEMs, have been identified. These domains are preformed structures ("physical entities") that compartmentalize proteins, lipids, and signaling molecules into multimolecular assemblies. In APCs, different microdomains containing immunoreceptors (MHC proteins, PRRs, integrins, among others) have been reported that are imperative for efficient pathogen recognition, the formation of the immunological synapse, and subsequent T cell activation. In addition, recent work has demonstrated that tetraspanin microdomains and lipid rafts are involved in BCR signaling and B cell activation. Research into the molecular mechanisms underlying membrane domain formation is fundamental to a comprehensive understanding of membrane-proximal signaling and APC function. This review will also discuss the advances in the microscopy field for the visualization of the plasma membrane, as well as the recent progress in targeting microdomains as novel, therapeutic approach for infectious and malignant diseases.

Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR

Energy Technology Data Exchange (ETDEWEB)

Tumulka, Franz [Goethe-University Frankfurt, Institute of Biochemistry, Biocenter (Germany); Roos, Christian; Loehr, Frank [Goethe-University Frankfurt, Institute of Biophysical Chemistry, Biocenter (Germany); Bock, Christoph [Goethe-University Frankfurt, Institute of Biochemistry, Biocenter (Germany); Bernhard, Frank; Doetsch, Volker [Goethe-University Frankfurt, Institute of Biophysical Chemistry, Biocenter (Germany); Abele, Rupert, E-mail: abele@em.uni-frankfurt.de [Goethe-University Frankfurt, Institute of Biochemistry, Biocenter (Germany)

2013-10-15

The ATP binding cassette transporter TAPL translocates cytosolic peptides into the lumen of lysosomes driven by the hydrolysis of ATP. Functionally, this transporter can be divided into coreTAPL, comprising the transport function, and an additional N-terminal transmembrane domain called TMD0, which is essential for lysosomal targeting and mediates the interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. To elucidate the structure of this unique domain, we developed protocols for the production of high quantities of cell-free expressed TMD0 by screening different N-terminal expression tags. Independently of the amino acid sequence, high expression was detected for AU-rich sequences in the first seven codons, decreasing the free energy of RNA secondary structure formation at translation initiation. Furthermore, avoiding NGG codons in the region of translation initiation demonstrated a positive effect on expression. For NMR studies, conditions were optimized for high solubilization efficiency, long-term stability, and high quality spectra. A most critical step was the careful exchange of the detergent used for solubilization by the detergent dihexanoylphosphatidylcholine. Several constructs of different size were tested in order to stabilize the fold of TMD0 as well as to reduce the conformation exchange. NMR spectra with sufficient resolution and homogeneity were finally obtained with a TMD0 derivative only modified by a C-terminal His{sub 10}-tag and containing a codon optimized AT-rich sequence.

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

Directory of Open Access Journals (Sweden)

Monica Salamone

Full Text Available In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4 and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs or Serine Integral Membrane Peptidases (SIMPs caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

Science.gov (United States)

Salamone, Monica; Carfì Pavia, Francesco; Ghersi, Giulio

2016-01-01

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

Science.gov (United States)

Salamone, Monica; Carfì Pavia, Francesco

2016-01-01

In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

Mechanisms of membrane binding of small GTPase K-Ras4B farnesylated hypervariable region.

Science.gov (United States)

Jang, Hyunbum; Abraham, Sherwin J; Chavan, Tanmay S; Hitchinson, Ben; Khavrutskii, Lyuba; Tarasova, Nadya I; Nussinov, Ruth; Gaponenko, Vadim

2015-04-10

K-Ras4B belongs to a family of small GTPases that regulates cell growth, differentiation and survival. K-ras is frequently mutated in cancer. K-Ras4B association with the plasma membrane through its farnesylated and positively charged C-terminal hypervariable region (HVR) is critical to its oncogenic function. However, the structural mechanisms of membrane association are not fully understood. Here, using confocal microscopy, surface plasmon resonance, and molecular dynamics simulations, we observed that K-Ras4B can be distributed in rigid and loosely packed membrane domains. Its membrane binding domain interaction with phospholipids is driven by membrane fluidity. The farnesyl group spontaneously inserts into the disordered lipid microdomains, whereas the rigid microdomains restrict the farnesyl group penetration. We speculate that the resulting farnesyl protrusion toward the cell interior allows oligomerization of the K-Ras4B membrane binding domain in rigid microdomains. Unlike other Ras isoforms, K-Ras4B HVR contains a single farnesyl modification and positively charged polylysine sequence. The high positive charge not only modulates specific HVR binding to anionic phospholipids but farnesyl membrane orientation. Phosphorylation of Ser-181 prohibits spontaneous farnesyl membrane insertion. The mechanism illuminates the roles of HVR modifications in K-Ras4B targeting microdomains of the plasma membrane and suggests an additional function for HVR in regulation of Ras signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

THE NANOSTRUCTURE OF ERYTHROCYTE MEMBRANES UNDER BLOOD INTOXICATION: AN ATOMIC FORCE MICROSCOPY STUDY

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V. A. Sergunova

2016-01-01

Full Text Available Background: The effects of toxins on nanostructure of blood cells are one of the key problems of biophysics and medicine. Erythrocyte morphology and membrane structure are recognized as the main parameters of blood quality. Therefore, analysis of membrane defects under toxin effects seems an urgent issue. Aim: To identify characteristic features and patterns of changes in membrane nanostructure under hemin intoxication and during extended storage of erythrocyte suspension. Materials and methods: The study was done in vitro in human whole blood with addition of hemin, аnd in erythrocyte suspension with a CPD blood preservative stored at 4 °С for 30 days. The nanostructure of erythrocyte membrane was assessed by atomic force microscopy. Results: Characteristic size of space periods between “granules” was from 120 to 200 nm. “Granule” numbers within a topological defect varied from 4 to 5 and to several dozens. Such domains arose virtually on all cells in erythrocyte suspension, as well as after hemin addition to the blood. An increase in hemin intoxication and an increase in a storage time were associated by increases in echinocyte numbers that subsequently transformed into spherical echinocytes. Both under hemin and during the storage of erythrocyte suspension for 9 to 12 days, a specific abnormality in nanostructure of erythrocyte membrane was observed: structural clusters, i.e., domains with granular structure, were formed. Conclusion: The experiments showed that both hemin and oxidative processes in the blood can specifically affect the nanostructure of erythrocyte membranes with formation of domains on their surface. The specific size of granular structures in the domains is from 100 to 200 nm that coincides with a  specific size of spectrin matrix. These results can be used in basic and applied medicine, in blood transfusion, for the analysis of a toxin effects in the human body. The biophysical mechanisms of domain

Isogeometric Shape Optimization of Vibrating Membranes

DEFF Research Database (Denmark)

Nguyen, Dang Manh; Evgrafov, Anton; Gersborg, Allan Roulund

2011-01-01

We consider a model problem of isogeometric shape optimization of vibrating membranes whose shapes are allowed to vary freely. The main obstacle we face is the need for robust and inexpensive extension of a B-spline parametrization from the boundary of a domain onto its interior, a task which has...... perform a number of numerical experiments with our isogeometric shape optimization algorithm and present smooth, optimized membrane shapes. Our conclusion is that isogeometric analysis fits well with shape optimization....

Effects of Lateral and Terminal Chains of X-Shaped Bolapolyphiles with Oligo(phenylene ethynylene Cores on Self-Assembly Behavior. Part 2: Domain Formation by Self-Assembly in Lipid Bilayer Membranes

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Stefan Werner

2017-09-01

Full Text Available Supramolecular self-assembly of membrane constituents within a phospholipid bilayer creates complex functional platforms in biological cells that operate in intracellular signaling, trafficking and membrane remodeling. Synthetic polyphilic compounds of macromolecular or small size can be incorporated into artificial phospholipid bilayers. Featuring three or four moieties of different philicities, they reach beyond ordinary amphiphilicity and open up avenues to new functions and interaction concepts. Here, we have incorporated a series of X-shaped bolapolyphiles into DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine bilayers of giant unilamellar vesicles. The bolapolyphiles consist of a rod-like oligo(phenylene ethynylene (OPE core, hydrophilic glycerol-based headgroups with or without oligo(ethylene oxide expansions at both ends and two lateral alkyl chains attached near the center of the OPE core. In the absence of DPPC and water, the compounds showed thermotropic liquid-crystalline behavior with a transition between polyphilic and amphiphilic assembly (see part 1 in this issue. In DPPC membranes, various trends in the domain morphologies were observed upon structure variations, which entailed branched alkyl chains of various sizes, alkyl chain semiperfluorination and size expansion of the headgroups. Observed effects on domain morphology are interpreted in the context of the bulk behavior (part 1 and of a model that was previously developed based on spectroscopic and physicochemical data.

Finsler Geometry Modeling of Phase Separation in Multi-Component Membranes

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Satoshi Usui

2016-08-01

Full Text Available A Finsler geometric surface model is studied as a coarse-grained model for membranes of three components, such as zwitterionic phospholipid (DOPC, lipid (DPPC and an organic molecule (cholesterol. To understand the phase separation of liquid-ordered (DPPC rich L o and liquid-disordered (DOPC rich L d , we introduce a binary variable σ ( = ± 1 into the triangulated surface model. We numerically determine that two circular and stripe domains appear on the surface. The dependence of the morphological change on the area fraction of L o is consistent with existing experimental results. This provides us with a clear understanding of the origin of the line tension energy, which has been used to understand these morphological changes in three-component membranes. In addition to these two circular and stripe domains, a raft-like domain and budding domain are also observed, and the several corresponding phase diagrams are obtained.

Effective Debye length in closed nanoscopic systems: a competition between two length scales.

Science.gov (United States)

Tessier, Frédéric; Slater, Gary W

2006-02-01

The Poisson-Boltzmann equation (PBE) is widely employed in fields where the thermal motion of free ions is relevant, in particular in situations involving electrolytes in the vicinity of charged surfaces. The applications of this non-linear differential equation usually concern open systems (in osmotic equilibrium with an electrolyte reservoir, a semi-grand canonical ensemble), while solutions for closed systems (where the number of ions is fixed, a canonical ensemble) are either not appropriately distinguished from the former or are dismissed as a numerical calculation exercise. We consider herein the PBE for a confined, symmetric, univalent electrolyte and quantify how, in addition to the Debye length, its solution also depends on a second length scale, which embodies the contribution of ions by the surface (which may be significant in high surface-to-volume ratio micro- or nanofluidic capillaries). We thus establish that there are four distinct regimes for such systems, corresponding to the limits of the two parameters. We also show how the PBE in this case can be formulated in a familiar way by simply replacing the traditional Debye length by an effective Debye length, the value of which is obtained numerically from conservation conditions. But we also show that a simple expression for the value of the effective Debye length, obtained within a crude approximation, remains accurate even as the system size is reduced to nanoscopic dimensions, and well beyond the validity range typically associated with the solution of the PBE.

Synchrotron nanoscopy imaging study of scalp hair in breast cancer patients and healthy individuals: Difference in medulla loss and cortical membrane enhancements.

Science.gov (United States)

Han, Sung-Mi; Chikawa, Jun-Ichi; Jeon, Jae-Kun; Hwang, Min-Young; Lim, Jun; Jeong, Young-Ju; Park, Sung-Hwan; Kim, Hong-Tae; Jheon, Sanghoon; Kim, Jong-Ki

2016-01-01

Nanoscopic synchrotron X-ray imaging was performed on scalp hair samples of patients with breast cancer and healthy individuals to investigate any structural differences as diagnostic tool. Hair strands were divided into 2-3 segments along the strands from root to tip, followed by imaging either in projection or in CT scanning with a monochromatic 6.78-keV X-ray using zone-plate optics with a resolving power of 60 nm. All the examined cancer hairs exhibited medulla loss with cancer stage-dependent pattern; complete loss, discontinuous or trace along the strands. In contrast, medullas were well retained without complete loss in the healthy hair. In the CT-scanned axial images, the cortical spindle compartments had no contrast in the healthy hair, but appeared hypointense in contrast to the surrounding hyperintense cortical membrane complex in the cancer hair. In conclusion, observation of medulla loss and cortical membrane enhancements in the hair strands of breast cancer patients demonstrated structural variations in the cancer hair, providing a new platform for further synchrotron X-ray imaging study of screening breast cancer patients. © 2015 Wiley Periodicals, Inc.

Uniform Structure of Eukaryotic Plasma Membrane: Lateral Domains in Plants

Czech Academy of Sciences Publication Activity Database

Malínská, Kateřina; Zažímalová, Eva

2011-01-01

Roč. 12, č. 2 (2011), s. 148-155 ISSN 1389-2037 R&D Projects: GA MŠk(CZ) LC06034 Institutional research plan: CEZ:AV0Z50380511 Keywords : Plasma membrane * microdomains * lateral segregation Subject RIV: ED - Physiology Impact factor: 2.886, year: 2011

Nonperturbative Renormalization Group Approach to Polymerized Membranes

Science.gov (United States)

Essafi, Karim; Kownacki, Jean-Philippe; Mouhanna, Dominique

2014-03-01

Membranes or membrane-like materials play an important role in many fields ranging from biology to physics. These systems form a very rich domain in statistical physics. The interplay between geometry and thermal fluctuations lead to exciting phases such flat, tubular and disordered flat phases. Roughly speaking, membranes can be divided into two group: fluid membranes in which the molecules are free to diffuse and thus no shear modulus. On the other hand, in polymerized membranes the connectivity is fixed which leads to elastic forces. This difference between fluid and polymerized membranes leads to a difference in their critical behaviour. For instance, fluid membranes are always crumpled, whereas polymerized membranes exhibit a phase transition between a crumpled phase and a flat phase. In this talk, I will focus only on polymerized phantom, i.e. non-self-avoiding, membranes. The critical behaviour of both isotropic and anisotropic polymerized membranes are studied using a nonperturbative renormalization group approach (NPRG). This allows for the investigation of the phase transitions and the low temperature flat phase in any internal dimension D and embedding d. Interestingly, graphene behaves just as a polymerized membrane in its flat phase.

The Origin of Nanoscopic Grooving on Vesicle Walls in Submarine Basaltic Glass: Implications for Nanotechnology

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Jason E. French

2009-01-01

Full Text Available Dendritic networks of nanoscopic grooves measuring 50–75 nm wide by <50 nm deep occur on the walls of vesicles in the glassy margins of mid-ocean ridge pillow basalts worldwide. Until now, their exact origin and significance have remained unclear. Here we document examples of such grooved patterns on vesicle walls in rocks from beneath the North Atlantic Ocean, and give a fluid mechanical explanation for how they formed. According to this model, individual nanogrooves represent frozen viscous fingers of magmatic fluid that were injected into a thin spheroidal shell of hot glass surrounding each vesicle. The driving mechanism for this process is provided by previous numerical predictions of tangential tensile stress around some vesicles in glassy rocks upon cooling through the glass transition. The self-assembling nature of the dendritic nanogrooves, their small size, and overall complexity in form, are interesting from the standpoint of exploring new applications in the field of nanotechnology. Replicating such structures in the laboratory would compete with state-of-the-art nanolithography techniques, both in terms of pattern complexity and size, which would be useful in the fabrication of a variety of grooved nanodevices. Dendritic nanogrooving in SiO2 glass might be employed in the manufacturing of integrated circuits.

Structural basis for different phosphoinositide specificities of the PX domains of sorting nexins regulating G-protein signaling.

Science.gov (United States)

Mas, Caroline; Norwood, Suzanne J; Bugarcic, Andrea; Kinna, Genevieve; Leneva, Natalya; Kovtun, Oleksiy; Ghai, Rajesh; Ona Yanez, Lorena E; Davis, Jasmine L; Teasdale, Rohan D; Collins, Brett M

2014-10-10

Sorting nexins (SNXs) or phox homology (PX) domain containing proteins are central regulators of cell trafficking and signaling. A subfamily of PX domain proteins possesses two unique PX-associated domains, as well as a regulator of G protein-coupled receptor signaling (RGS) domain that attenuates Gαs-coupled G protein-coupled receptor signaling. Here we delineate the structural organization of these RGS-PX proteins, revealing a protein family with a modular architecture that is conserved in all eukaryotes. The one exception to this is mammalian SNX19, which lacks the typical RGS structure but preserves all other domains. The PX domain is a sensor of membrane phosphoinositide lipids and we find that specific sequence alterations in the PX domains of the mammalian RGS-PX proteins, SNX13, SNX14, SNX19, and SNX25, confer differential phosphoinositide binding preferences. Although SNX13 and SNX19 PX domains bind the early endosomal lipid phosphatidylinositol 3-phosphate, SNX14 shows no membrane binding at all. Crystal structures of the SNX19 and SNX14 PX domains reveal key differences, with alterations in SNX14 leading to closure of the binding pocket to prevent phosphoinositide association. Our findings suggest a role for alternative membrane interactions in spatial control of RGS-PX proteins in cell signaling and trafficking. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The molecular architecture of the chloroplast thylakoid membrane

Energy Technology Data Exchange (ETDEWEB)

Stefansson, H.

1996-08-01

Non-detergent procedure for isolation of sub-thylakoid vesicle populations derived from different structural domains of the chloroplast thylakoid membrane has been developed. Sub-thylakoid vesicles representing the grana, grana core, stroma lamellae, and the grana margins have been isolated and their protein composition has been investigated. Furthermore a novel non-detergent procedure for investigating the pigment composition of photosynthetic complexes located in the different structural domains has been developed. This procedure circumvents selective extractions, an perturbing effect often combined with detergent isolations of membrane bound protein complexes. The fractionation experiments show that the NADPH dehydrogenase, suggested to operate as NADPH or ferredoxin-plastoquinone oxidoreductase in cyclic electron transport around photosystem I, is stoichiometrically depleted on photosystem I basis in the grana domain. The fractionation studies are consistent with the model of the thylakoid membrane where the photosystems in the grana are operating in a linear electron transport whereas the site of cyclic electron transport is in the stroma lamellae. It is suggested that partial destacking of grana, as a result of light-induced protein phosphorylation, may promote the exposure of the granal photosystem I centers to the chloroplast stroma and thereby enhance their participation in cyclic electron transport activity. 146 refs, 18 figs

The actin homologue MreB organizes the bacterial cell membrane

OpenAIRE

Strahl, Henrik; Bürmann, Frank; Hamoen, Leendert W.

2014-01-01

The eukaryotic cortical actin cytoskeleton creates specific lipid domains, including lipid rafts, which determine the distribution of many membrane proteins. Here we show that the bacterial actin homologue MreB displays a comparable activity. MreB forms membrane-associated filaments that coordinate bacterial cell wall synthesis. We noticed that the MreB cytoskeleton influences fluorescent staining of the cytoplasmic membrane. Detailed analyses combining an array of mutants, using specific lip...

Fatty acid profiles from the plasma membrane and detergent resistant membranes of two plant species.

Science.gov (United States)

Carmona-Salazar, Laura; El Hafidi, Mohammed; Gutiérrez-Nájera, Nora; Noyola-Martínez, Liliana; González-Solís, Ariadna; Gavilanes-Ruíz, Marina

2015-01-01

It is essential to establish the composition of the plant plasma membrane in order to understand its organization and behavior under continually changing environments. Knowledge of the lipid phase, in particular the fatty acid (FA) complex repertoire, is important since FAs determine many of the physical-chemical membrane properties. FAs are constituents of the membrane glycerolipid and sphingolipid backbones and can also be linked to some sterols. In addition, FAs are components of complex lipids that can constitute membrane micro-domains, and the use of detergent-resistant membranes is a common approach to study their composition. The diversity and cellular allocation of the membrane lipids containing FAs are very diverse and the approaches to analyze them provide only general information. In this work, a detailed FA analysis was performed using highly purified plasma membranes from bean leaves and germinating maize embryos and their respective detergent-resistant membrane preparations. The analyses showed the presence of a significant amount of very long chain FAs (containing 28C, 30C and 32C), in both plasma membrane preparations from bean and maize, that have not been previously reported. Herein is demonstrated that a significant enrichment of very long chain saturated FAs and saturated FAs can occur in detergent-resistant membrane preparations, as compared to the plasma membranes from both plant species. Considering that a thorough analysis of FAs is rarely performed in purified plasma membranes and detergent-resistant membranes, this work provides qualitative and quantitative evidence on the contributions of the length and saturation of FAs to the organization of the plant plasma membrane and detergent-resistant membranes. Copyright © 2014 Elsevier Ltd. All rights reserved.

Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains

DEFF Research Database (Denmark)

Willumsen, B M; Papageorge, A G; Hubbert, N

1985-01-01

or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences...... that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids...... that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor...

Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases.

Science.gov (United States)

Jadwin, Joshua A; Oh, Dongmyung; Curran, Timothy G; Ogiue-Ikeda, Mari; Jia, Lin; White, Forest M; Machida, Kazuya; Yu, Ji; Mayer, Bruce J

2016-04-12

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.

Hydrophobic mismatch triggering texture defects in membrane gel domains

DEFF Research Database (Denmark)

Dreier, J.; Brewer, J.R.; Simonsen, Adam Cohen

2013-01-01

higher mismatch values correlate with a vortex-type texture. The defect pattern created during early growth persists in larger domains, and a minimal model incorporating the anisotropic line tension and the vortex energy can rationalize this finding. The results suggest that the lipid composition...

A cataract-causing connexin 50 mutant is mislocalized to the ER due to loss of the fourth transmembrane domain and cytoplasmic domain.

Science.gov (United States)

Somaraju Chalasani, Madhavi Latha; Muppirala, Madhavi; G Ponnam, Surya Prakash; Kannabiran, Chitra; Swarup, Ghanshyam

2013-01-01

Mutations in the eye lens gap junction protein connexin 50 cause cataract. Earlier we identified a frameshift mutant of connexin 50 (c.670insA; p.Thr203AsnfsX47) in a family with autosomal recessive cataract. The mutant protein is smaller and contains 46 aberrant amino acids at the C-terminus after amino acid 202. Here, we have analysed this frameshift mutant and observed that it localized to the endoplasmic reticulum (ER) but not in the plasma membrane. Moreover, overexpression of the mutant resulted in disintegration of the ER-Golgi intermediate compartment (ERGIC), reduction in the level of ERGIC-53 protein and breakdown of the Golgi in many cells. Overexpression of the frameshift mutant partially inhibited the transport of wild type connexin 50 to the plasma membrane. A deletion mutant lacking the aberrant sequence showed predominant localization in the ER and inhibited anterograde protein transport suggesting, therefore, that the aberrant sequence is not responsible for improper localization of the frameshift mutant. Further deletion analysis showed that the fourth transmembrane domain and a membrane proximal region (231-294 amino acids) of the cytoplasmic domain are needed for transport from the ER and localization to the plasma membrane. Our results show that a frameshift mutant of connexin 50 mislocalizes to the ER and causes disintegration of the ERGIC and Golgi. We have also identified a sequence of connexin 50 crucial for transport from the ER and localization to the plasma membrane.

Atomic force microscopy on domains in biological model membranes

NARCIS (Netherlands)

Rinia, H.A.

2001-01-01

This thesis describes the preparation and imaging of supported lipid bilayers, which can be regarded as biological modelmembranes, in the light of the formation of domains. The bilayers were prepared with either the Langmuir-Blodgett method, or with vesicle fusion. They were imaged with Atomic Force

Resolution of G(s)alpha and G(q)alpha/G(11)alpha proteins in membrane domains by two-dimensional electrophoresis: the effect of long-term agonist stimulation.

Science.gov (United States)

Matousek, P; Novotný, J; Svoboda, P

2004-01-01

Low-density membrane-domain fractions were prepared from S49 lymphoma cells and clone e2m11 of HEK293 cells expressing a large number of thyrotropin-releasing hormone receptor (TRH-R) and G(11)alpha by flotation on sucrose density gradients. The intact cell structure was broken by detergent-extraction, alkaline-treatment or drastic homogenization. Three types of low-density membranes were resolved by two-dimensional electrophoresis and analyzed for G(s)alpha (S49) or G(q)alpha/G11) (e2m11) content. Four individual immunoblot signals of Gsalpha protein were identified in S49 lymphoma cells indicating complete resolution of the long G(s)alpha L+/-ser and short G(s)alpha S+/-ser variants of G(s)alpha. All these were diminished by prolonged agonist (isoprenaline) stimulation. In e2m11-HEK cells, five different immunoblot signals were detected indicating post-translational modification of G proteins of G(q)alpha/G(11)alpha family. The two major spots corresponding to exogenously (over)expressed G(11)alpha and endogenous G(q)alpha were reduced; the minor spots diminished by hormonal stimulation. Parallel analysis by silver staining of the total protein content indicated that no major changes in protein composition occurred under these conditions. Our data thus indicate that agonist-stimulation of target cells results in down-regulation of all different members of G(s) and G(q)/G(11) families. This agonist-specific effect may be demonstrated in crude membrane as well as domain/raft preparations and it is not accompanied by changes in overall protein composition.

Dissect Kif5b in nuclear positioning during myogenesis: The light chain binding domain and the autoinhibitory peptide are both indispensable

Energy Technology Data Exchange (ETDEWEB)

Wang, Zai, E-mail: wangzai81@hotmail.com [Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam (Hong Kong); Institute of Clinical Medical Sciences, China–Japan Friendship Hospital, Beijing (China); Xue, Wenqian; Li, Xiuling; Lin, Raozhou [Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam (Hong Kong); Cui, Ju [Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam (Hong Kong); Beijing Institute of Geriatrics, Beijing Hospital, Ministry of Health (China); Huang, Jian-Dong, E-mail: jdhuang@hku.hk [Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam (Hong Kong)

2013-03-08

Highlights: ► Kif5b localizes at myonuclear membrane and is responsible for nuclear dispersion. ► Kif5b stalk/tail domain contains signal for nuclear membrane targeting. ► Kif5b stalk/tail domain directly binds to a nesprin 4 in vitro. ► KLC binding domain and autoinhibitory peptide are both functionally indispensable. -- Abstract: The microtubule motor kinesin-1 is responsible for the nuclear positioning during myogenesis. Here we show that the coiled-coil stalk/tail domain containing the kinesin light chain (KLC) binding sites targets to the perinuclear region like endogenous Kif5b, while the globular tail domain cannot. To investigate which fragments of kinesin heavy chain (Kif5b) is responsible for the myonuclear positioning, we transfect Kif5b expression constructs into Kif5b deficient myoblasts and test their ability to rescue the myonuclear phenotype. We find that the KLC binding domain and the autoinhibitory peptide in the globular tail region are both indispensable for the nuclear membrane localization of Kif5b and the kinesin-1-mediated myonuclear positioning. These results suggest that while the KLC binding domain may directly targets Kif5b to the myonuclear membrane, the autoinhibitory peptide may play an indirect role in regulating the kinesin-1-mediated myonuclear positioning.

Peripheral Protein Unfolding Drives Membrane Bending.

Science.gov (United States)

Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian

2018-06-20

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

Effect of spatial inhomogeneities on the membrane surface on receptor dimerization and signal initiation

Directory of Open Access Journals (Sweden)

Romica Kerketta

2016-08-01

Full Text Available Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as clusters by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones (domains for the two receptor species. The in silico model is parameterized and validated using data from single particle tracking experiments. We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems.

Pleckstrin Homology Domain Diffusion in Dictyostelium Cytoplasm Studied Using Fluorescence Correlation Spectroscopy

NARCIS (Netherlands)

Engel, Ruchira; Hink, Mark A.; Bosgraaf, Leonard; Haastert, Peter J.M. van; Visser, Antonie J.W.G.

2004-01-01

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

NARCIS (Netherlands)

Ruchira, A.; Hink, M.A.; Bosgraaf, L.; Haastert, van P.J.M.; Visser, A.J.W.G.

2004-01-01

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC

NMR structural studies of peptides and proteins in membranes

Energy Technology Data Exchange (ETDEWEB)

Opella, S J [Pennsylvania Univ., Philadelphia, PA (United States). Dept. of Chemistry

1994-12-31

The use of NMR methodology in structural studies is described as applicable to larger proteins, considering that the majority of membrane proteins is constructed from a limited repertoire of structural and dynamic elements. The membrane associated domains of these proteins are made up of long hydrophobic membrane spanning helices, shorter amphipathic bridging helices in the plane of the bilayer, connecting loops with varying degrees of mobility, and mobile N- and C- terminal sections. NMR studies have been successful in identifying all of these elements and their orientations relative to each other and the membrane bilayer 19 refs., 9 figs.

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy.

Science.gov (United States)

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM).

Phase behavior of multicomponent membranes: Experimental and computational techniques

DEFF Research Database (Denmark)

Bagatolli, Luis; Kumar, P.B. Sunil

2009-01-01

Recent developments in biology seems to indicate that the Fluid Mosaic model of membrane proposed by Singer and Nicolson, with lipid bilayer functioning only as medium to support protein machinery, may be too simple to be realistic. Many protein functions are now known to depend on the compositio....... This review includes basic foundations on membrane model systems and experimental approaches applied in the membrane research area, stressing on recent advances in the experimental and computational techniques....... membranes. Current increase in interest in the domain formation in multicomponent membranes also stems from the experiments demonstrating liquid ordered-liquid disordered coexistence in mixtures of lipids and cholesterol and the success of several computational models in predicting their behavior...

Protein transport across and into cell membranes in bacteria and archaea

NARCIS (Netherlands)

Yuan, Jijun; Zweers, Jessica C.; van Dijl, Jan Maarten; Dalbey, Ross E.

In the three domains of life, the Sec, YidC/Oxa1, and Tat translocases play important roles in protein translocation across membranes and membrane protein insertion. While extensive studies have been performed on the endoplasmic reticular and Escherichia coli systems, far fewer studies have been

Intermolecular detergent-membrane protein noes for the characterization of the dynamics of membrane protein-detergent complexes.

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Eichmann, Cédric; Orts, Julien; Tzitzilonis, Christos; Vögeli, Beat; Smrt, Sean; Lorieau, Justin; Riek, Roland

2014-12-11

The interaction between membrane proteins and lipids or lipid mimetics such as detergents is key for the three-dimensional structure and dynamics of membrane proteins. In NMR-based structural studies of membrane proteins, qualitative analysis of intermolecular nuclear Overhauser enhancements (NOEs) or paramagnetic resonance enhancement are used in general to identify the transmembrane segments of a membrane protein. Here, we employed a quantitative characterization of intermolecular NOEs between (1)H of the detergent and (1)H(N) of (2)H-perdeuterated, (15)N-labeled α-helical membrane protein-detergent complexes following the exact NOE (eNOE) approach. Structural considerations suggest that these intermolecular NOEs should show a helical-wheel-type behavior along a transmembrane helix or a membrane-attached helix within a membrane protein as experimentally demonstrated for the complete influenza hemagglutinin fusion domain HAfp23. The partial absence of such a NOE pattern along the amino acid sequence as shown for a truncated variant of HAfp23 and for the Escherichia coli inner membrane protein YidH indicates the presence of large tertiary structure fluctuations such as an opening between helices or the presence of large rotational dynamics of the helices. Detergent-protein NOEs thus appear to be a straightforward probe for a qualitative characterization of structural and dynamical properties of membrane proteins embedded in detergent micelles.

Analysis of Nanodomain Composition in High-Impact Polypropylene by Atomic Force Microscopy-Infrared.

Science.gov (United States)

Tang, Fuguang; Bao, Peite; Su, Zhaohui

2016-05-03

In this paper, compositions of nanodomains in a commercial high-impact polypropylene (HIPP) were investigated by an atomic force microscopy-infrared (AFM-IR) technique. An AFM-IR quantitative analysis method was established for the first time, which was then employed to analyze the polyethylene content in the nanoscopic domains of the rubber particles dispersed in the polypropylene matrix. It was found that the polyethylene content in the matrix was close to zero and was high in the rubbery intermediate layers, both as expected. However, the major component of the rigid cores of the rubber particles was found to be polypropylene rather than polyethylene, contrary to what was previously believed. The finding provides new insight into the complicated structure of HIPPs, and the AFM-IR quantitative method reported here offers a useful tool for assessing compositions of nanoscopic domains in complex polymeric systems.

Endocytosis of GPI-linked membrane folate receptor-alpha.

Science.gov (United States)

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

1996-01-01

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

Finding a needle in a haystack: the role of electrostatics in target lipid recognition by PH domains.

Directory of Open Access Journals (Sweden)

Craig N Lumb

Full Text Available Interactions between protein domains and lipid molecules play key roles in controlling cell membrane signalling and trafficking. The pleckstrin homology (PH domain is one of the most widespread, binding specifically to phosphatidylinositol phosphates (PIPs in cell membranes. PH domains must locate specific PIPs in the presence of a background of approximately 20% anionic lipids within the cytoplasmic leaflet of the plasma membrane. We investigate the mechanism of such recognition via a multiscale procedure combining Brownian dynamics (BD and molecular dynamics (MD simulations of the GRP1 PH domain interacting with phosphatidylinositol (3,4,5-trisphosphate (PI(3,4,5P₃. The interaction of GRP1-PH with PI(3,4,5P₃ in a zwitterionic bilayer is compared with the interaction in bilayers containing different levels of anionic 'decoy' lipids. BD simulations reveal both translational and orientational electrostatic steering of the PH domain towards the PI(3,4,5P₃-containing anionic bilayer surface. There is a payoff between non-PIP anionic lipids attracting the PH domain to the bilayer surface in a favourable orientation and their role as 'decoys', disrupting the interaction of GRP1-PH with the PI(3,4,5P₃ molecule. Significantly, approximately 20% anionic lipid in the cytoplasmic leaflet of the bilayer is nearly optimal to both enhance orientational steering and to localise GRP1-PH proximal to the surface of the membrane without sacrificing its ability to locate PI(3,4,5P₃ within the bilayer plane. Subsequent MD simulations reveal binding to PI(3,4,5P₃, forming protein-phosphate contacts comparable to those in X-ray structures. These studies demonstrate a computational framework which addresses lipid recognition within a cell membrane environment, offering a link between structural and cell biological characterisation.

Combinatorial mutagenesis of the voltage-sensing domain enables the optical resolution of action potentials firing at 60 Hz by a genetically encoded fluorescent sensor of membrane potential.

Science.gov (United States)

Piao, Hong Hua; Rajakumar, Dhanarajan; Kang, Bok Eum; Kim, Eun Ha; Baker, Bradley J

2015-01-07

ArcLight is a genetically encoded fluorescent voltage sensor using the voltage-sensing domain of the voltage-sensing phosphatase from Ciona intestinalis that gives a large but slow-responding optical signal in response to changes in membrane potential (Jin et al., 2012). Fluorescent voltage sensors using the voltage-sensing domain from other species give faster yet weaker optical signals (Baker et al., 2012; Han et al., 2013). Sequence alignment of voltage-sensing phosphatases from different species revealed conserved polar and charged residues at 7 aa intervals in the S1-S3 transmembrane segments of the voltage-sensing domain, suggesting potential coil-coil interactions. The contribution of these residues to the voltage-induced optical signal was tested using a cassette mutagenesis screen by flanking each transmembrane segment with unique restriction sites to allow for the testing of individual mutations in each transmembrane segment, as well as combinations in all four transmembrane segments. Addition of a counter charge in S2 improved the kinetics of the optical response. A double mutation in the S4 domain dramatically reduced the slow component of the optical signal seen in ArcLight. Combining that double S4 mutant with the mutation in the S2 domain yielded a probe with kinetics voltage-sensing domain could potentially lead to fluorescent sensors capable of optically resolving neuronal inhibition and subthreshold synaptic activity. Copyright © 2015 the authors 0270-6474/15/350372-15$15.00/0.

Endophilin-A1 BAR domain interaction with arachidonyl CoA.

Science.gov (United States)

Petoukhov, Maxim V; Weissenhorn, Winfried; Svergun, Dmitri I

2014-01-01

Endophilin-A1 belongs to the family of BAR domain containing proteins that catalyze membrane remodeling processes via sensing, inducing and stabilizing membrane curvature. We show that the BAR domain of endophilin-A1 binds arachidonic acid and molds its coenzyme A (CoA) activated form, arachidonyl-CoA into a defined structure. We studied low resolution structures of endophilin-A1-BAR and its complex with arachidonyl-CoA in solution using synchrotron small-angle X-ray scattering (SAXS). The free endophilin-A1-BAR domain is shown to be dimeric at lower concentrations but builds tetramers and higher order complexes with increasing concentrations. Extensive titration SAXS studies revealed that the BAR domain produces a homogenous complex with the lipid micelles. The structural model of the complexes revealed two arachidonyl-CoA micelles bound to the distal arms of an endophilin-A1-BAR dimer. Intriguingly, the radius of the bound micelles significantly decreases compared to that of the free micelles, and this structural result may provide hints on the potential biological relevance of the endophilin-A1-BAR interaction with arachidonyl CoA.

SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins.

Science.gov (United States)

Koch, C A; Anderson, D; Moran, M F; Ellis, C; Pawson, T

1991-05-03

Src homology (SH) regions 2 and 3 are noncatalytic domains that are conserved among a series of cytoplasmic signaling proteins regulated by receptor protein-tyrosine kinases, including phospholipase C-gamma, Ras GTPase (guanosine triphosphatase)-activating protein, and Src-like tyrosine kinases. The SH2 domains of these signaling proteins bind tyrosine phosphorylated polypeptides, implicated in normal signaling and cellular transformation. Tyrosine phosphorylation acts as a switch to induce the binding of SH2 domains, thereby mediating the formation of heteromeric protein complexes at or near the plasma membrane. The formation of these complexes is likely to control the activation of signal transduction pathways by tyrosine kinases. The SH3 domain is a distinct motif that, together with SH2, may modulate interactions with the cytoskeleton and membrane. Some signaling and transforming proteins contain SH2 and SH3 domains unattached to any known catalytic element. These noncatalytic proteins may serve as adaptors to link tyrosine kinases to specific target proteins. These observations suggest that SH2 and SH3 domains participate in the control of intracellular responses to growth factor stimulation.

Lateral Organization of Influenza Virus Proteins in the Budozone Region of the Plasma Membrane.

Science.gov (United States)

Leser, George P; Lamb, Robert A

2017-05-01

Influenza virus assembles and buds at the plasma membrane of virus-infected cells. The viral proteins assemble at the same site on the plasma membrane for budding to occur. This involves a complex web of interactions among viral proteins. Some proteins, like hemagglutinin (HA), NA, and M2, are integral membrane proteins. M1 is peripherally membrane associated, whereas NP associates with viral RNA to form an RNP complex that associates with the cytoplasmic face of the plasma membrane. Furthermore, HA and NP have been shown to be concentrated in cholesterol-rich membrane raft domains, whereas M2, although containing a cholesterol binding motif, is not raft associated. Here we identify viral proteins in planar sheets of plasma membrane using immunogold staining. The distribution of these proteins was examined individually and pairwise by using the Ripley K function, a type of nearest-neighbor analysis. Individually, HA, NA, M1, M2, and NP were shown to self-associate in or on the plasma membrane. HA and M2 are strongly coclustered in the plasma membrane; however, in the case of NA and M2, clustering depends upon the expression system used. Despite both proteins being raft resident, HA and NA occupy distinct but adjacent membrane domains. M2 and M1 strongly cocluster, but the association of M1 with HA or NA is dependent upon the means of expression. The presence of HA and NP at the site of budding depends upon the coexpression of other viral proteins. Similarly, M2 and NP occupy separate compartments, but an association can be bridged by the coexpression of M1. IMPORTANCE The complement of influenza virus proteins necessary for the budding of progeny virions needs to accumulate at budozones. This is complicated by HA and NA residing in lipid raft-like domains, whereas M2, although an integral membrane protein, is not raft associated. Other necessary protein components such as M1 and NP are peripherally associated with the membrane. Our data define spatial relationships

Functional Independence and Interdependence of the Src Homology Domains of Phospholipase C-γ1 in B-Cell Receptor Signal Transduction

Science.gov (United States)

DeBell, Karen E.; Stoica, Bogdan A.; Verí, Maria-Concetta; Di Baldassarre, Angela; Miscia, Sebastiano; Graham, Laurie J.; Rellahan, Barbara L.; Ishiai, Masamichi; Kurosaki, Tomohiro; Bonvini, Ezio

1999-01-01

B-cell receptor (BCR)-induced activation of phospholipase C-γ1 (PLCγ1) and PLCγ2 is crucial for B-cell function. While several signaling molecules have been implicated in PLCγ activation, the mechanism coupling PLCγ to the BCR remains undefined. The role of PLCγ1 SH2 and SH3 domains at different steps of BCR-induced PLCγ1 activation was examined by reconstitution in a PLCγ-negative B-cell line. PLCγ1 membrane translocation required a functional SH2 N-terminal [SH2(N)] domain, was decreased by mutation of the SH3 domain, but was unaffected by mutation of the SH2(C) domain. Tyrosine phosphorylation did not require the SH2(C) or SH3 domains but depended exclusively on a functional SH2(N) domain, which mediated the association of PLCγ1 with the adapter protein, BLNK. Forcing PLCγ1 to the membrane via a myristoylation signal did not bypass the SH2(N) domain requirement for phosphorylation, indicating that the phosphorylation mediated by this domain is not due to membrane anchoring alone. Mutation of the SH2(N) or the SH2(C) domain abrogated BCR-stimulated phosphoinositide hydrolysis and signaling events, while mutation of the SH3 domain partially decreased signaling. PLCγ1 SH domains, therefore, have interrelated but distinct roles in BCR-induced PLCγ1 activation. PMID:10523627

Remodeling of the postsynaptic plasma membrane during neural development.

Science.gov (United States)

Tulodziecka, Karolina; Diaz-Rohrer, Barbara B; Farley, Madeline M; Chan, Robin B; Di Paolo, Gilbert; Levental, Kandice R; Waxham, M Neal; Levental, Ilya

2016-11-07

Neuronal synapses are the fundamental units of neural signal transduction and must maintain exquisite signal fidelity while also accommodating the plasticity that underlies learning and development. To achieve these goals, the molecular composition and spatial organization of synaptic terminals must be tightly regulated; however, little is known about the regulation of lipid composition and organization in synaptic membranes. Here we quantify the comprehensive lipidome of rat synaptic membranes during postnatal development and observe dramatic developmental lipidomic remodeling during the first 60 postnatal days, including progressive accumulation of cholesterol, plasmalogens, and sphingolipids. Further analysis of membranes associated with isolated postsynaptic densities (PSDs) suggests the PSD-associated postsynaptic plasma membrane (PSD-PM) as one specific location of synaptic remodeling. We analyze the biophysical consequences of developmental remodeling in reconstituted synaptic membranes and observe remarkably stable microdomains, with the stability of domains increasing with developmental age. We rationalize the developmental accumulation of microdomain-forming lipids in synapses by proposing a mechanism by which palmitoylation of the immobilized scaffold protein PSD-95 nucleates domains at the postsynaptic plasma membrane. These results reveal developmental changes in lipid composition and palmitoylation that facilitate the formation of postsynaptic membrane microdomains, which may serve key roles in the function of the neuronal synapse. © 2016 Tulodziecka et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Structural refinement of the hERG1 pore and voltage-sensing domains with ROSETTA-membrane and molecular dynamics simulations.

Science.gov (United States)

Subbotina, Julia; Yarov-Yarovoy, Vladimir; Lees-Miller, James; Durdagi, Serdar; Guo, Jiqing; Duff, Henry J; Noskov, Sergei Yu

2010-11-01

The hERG1 gene (Kv11.1) encodes a voltage-gated potassium channel. Mutations in this gene lead to one form of the Long QT Syndrome (LQTS) in humans. Promiscuous binding of drugs to hERG1 is known to alter the structure/function of the channel leading to an acquired form of the LQTS. Expectably, creation and validation of reliable 3D model of the channel have been a key target in molecular cardiology and pharmacology for the last decade. Although many models were built, they all were limited to pore domain. In this work, a full model of the hERG1 channel is developed which includes all transmembrane segments. We tested a template-driven de-novo design with ROSETTA-membrane modeling using side-chain placements optimized by subsequent molecular dynamics (MD) simulations. Although backbone templates for the homology modeled parts of the pore and voltage sensors were based on the available structures of KvAP, Kv1.2 and Kv1.2-Kv2.1 chimera channels, the missing parts are modeled de-novo. The impact of several alignments on the structure of the S4 helix in the voltage-sensing domain was also tested. Herein, final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. These structural elements include salt bridges and close contacts in the voltage-sensor domain; and the topology of the extracellular S5-pore linker compared with that established by toxin foot-printing and nuclear magnetic resonance studies. Implications of the refined hERG1 model to binding of blockers and channels activators (potent new ligands for channel activations) are discussed. © 2010 Wiley-Liss, Inc.

Secretory Vesicle Priming by CAPS Is Independent of Its SNARE-Binding MUN Domain

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Cuc Quynh Nguyen Truong

2014-11-01

Full Text Available Priming of secretory vesicles is a prerequisite for their Ca2+-dependent fusion with the plasma membrane. The key vesicle priming proteins, Munc13s and CAPSs, are thought to mediate vesicle priming by regulating the conformation of the t-SNARE syntaxin, thereby facilitating SNARE complex assembly. Munc13s execute their priming function through their MUN domain. Given that the MUN domain of Ca2+-dependent activator protein for secretion (CAPS also binds syntaxin, it was assumed that CAPSs prime vesicles through the same mechanism as Munc13s. We studied naturally occurring splice variants of CAPS2 in CAPS1/CAPS2-deficient cells and found that CAPS2 primes vesicles independently of its MUN domain. Instead, the pleckstrin homology domain of CAPS2 seemingly is essential for its priming function. Our findings indicate a priming mode for secretory vesicles. This process apparently requires membrane phospholipids, does not involve the binding or direct conformational regulation of syntaxin by MUN domains of CAPSs, and is therefore not redundant with Munc13 action.

Membrane Curvature and Lipid Composition Synergize To Regulate N-Ras Anchor Recruitment

DEFF Research Database (Denmark)

Larsen, Jannik B.; Kennard, Celeste; Pedersen, Søren L.

2017-01-01

Proteins anchored to membranes through covalently linked fatty acids and/or isoprenoid groups play crucial roles in all forms of life. Sorting and trafficking of lipidated proteins has traditionally been discussed in the context of partitioning to membrane domains of different lipid composition. We...

Poly-benzyl domains grown on porous silicon and their I-V rectification

International Nuclear Information System (INIS)

Chao Jie; Han Huanmei; Xia Bing; Ba Long; Liu Hongbo; Xiao Shoujun

2007-01-01

Microwave-irradiated polymerization of benzyl chloride and triphenyl chloromethane on hydride-terminated porous silicon (PS) was achieved through the use of Zn powder as a catalyst. Transmission infrared Fourier-transform spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) analyses confirmed the poly-benzyl membranes grafted on PS. Topographical images by AFM revealed crystal-like domains rather than homogenous monolayers on the surface. The current-voltage measurements in nano-scale by current sensing atomic force microscopy (CS-AFM) showed the rectification behavior of this polymer membrane. Finally, mechanism of a radical initiation on the surface and a following Friedel-Crafts alkylation was proposed for the covalent assembly of poly-benzyl domains

Pyrolytic carbon membranes containing silica: morphological approach on gas transport behavior

Science.gov (United States)

Park, Ho Bum; Lee, Sun Yong; Lee, Young Moo

2005-04-01

Pyrolytic carbon membrane containing silica (C-SiO 2) is a new-class material for gas separation, and in the present work we will deal with it in view of the morphological changes arising from the difference in the molecular structure of the polymeric precursors. The silica embedded carbon membranes were fabricated by a predetermined pyrolysis step using imide-siloxane copolymers (PISs) that was synthesized from benzophenone tetracarboxylic dianhydrides (BTDA), 4,4'-oxydianiline (ODA), and amine-terminated polydimethylsiloxane (PDMS). To induce different morphologies at the same chemical composition, the copolymers were prepared using one-step (preferentially a random segmented copolymer) sand two-step polymerization (a block segmented copolymer) methods. The polymeric precursors and their pyrolytic C-SiO 2 membranes were analyzed using thermal analysis, atomic force microscopy, and transmission electron microscopy, etc. It was found that the C-SiO 2 membrane derived from the random PIS copolymer showed a micro-structure containing small well-dispersed silica domains, whereas the C-SiO 2 membrane from the block PIS copolymer exhibited a micro-structure containing large silica domains in the continuous carbon matrix. Eventually, the gas transport through these C-SiO 2 membranes was significantly affected by the morphological changes of the polymeric precursors.

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Energy Technology Data Exchange (ETDEWEB)

Dick, Robert A.; Datta, Siddhartha A.K.; Nanda, Hirsh; Fang, Xianyang; Wen, Yi; Barros, Marilia; Wang, Yun-Xing; Rein, Alan; Vogt, Volker M. (NCI); (Cornell); (CM); (NIST)

2016-05-06

Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our

Phase Behavior and Domain Size in Sphingomyelin-Containing Lipid Bilayers

Science.gov (United States)

Petruzielo, Robin S.; Heberle, Frederick A.; Drazba, Paul; Katsaras, John; Feigenson, Gerald W.

2013-01-01

Membrane raft size measurements are crucial to understanding the stability and functionality of rafts in cells. The challenge of accurately measuring raft size is evidenced by the disparate reports of domain sizes, which range from nanometers to microns for the ternary model membrane system sphingomyelin (SM)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). Using Förster resonance energy transfer (FRET) and differential scanning calorimetry (DSC), we established phase diagrams for porcine brain SM (bSM)/dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and bSM/POPC/Chol at 15 and 25°C. By combining two techniques with different spatial sensitivities, namely FRET and small-angle neutron scattering (SANS), we have significantly narrowed the uncertainty in domain size estimates for bSM/POPC/Chol mixtures. Compositional trends in FRET data revealed coexisting domains at 15 and 25°C for both mixtures, while SANS measurements detected no domain formation for bSM/POPC/Chol. Together these results indicate that liquid domains in bSM/POPC/Chol are between 2 and 7 nm in radius at 25°C: that is, domains must be on the order of the 2–6 nm Förster distance of the FRET probes, but smaller than the ~7 nm minimum cluster size detectable with SANS. However, for palmitoyl SM (PSM)/POPC/Chol at a similar composition, SANS detected coexisting liquid domains. This increase in domain size upon replacing the natural SM component (which consists of a mixture of chain lengths) with synthetic PSM, suggests a role for SM chain length in modulating raft size in vivo. PMID:23337475

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability.

Science.gov (United States)

Smith, Alyson S; Nowak, Roberta B; Zhou, Sitong; Giannetto, Michael; Gokhin, David S; Papoin, Julien; Ghiran, Ionita C; Blanc, Lionel; Wan, Jiandi; Fowler, Velia M

2018-05-08

The biconcave disk shape and deformability of mammalian RBCs rely on the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network of RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by superresolution fluorescence microscopy. MgATP disrupts NMIIA association with the membrane skeleton, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical properties. In addition, the phosphorylation of the RBC NMIIA heavy and light chains in vivo indicates active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disk cell shape. As structures similar to the RBC membrane skeleton exist in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.

Structuring detergents for extracting and stabilizing functional membrane proteins.

Directory of Open Access Journals (Sweden)