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Sample records for integral membrane domain

  1. Porous Nanocomposites with Integrated Internal Domains: Application to Separation Membranes

    PubMed Central

    Li, Wenle; Walz, John Y.

    2014-01-01

    Asymmetric membranes with layered structure have made significant achievements due to their balanced properties and multi-functionalities that come from a combination of multiple layers. However, issues such as delamination and substructure resistance are generated by the intrinsic layered structure. Here, we present a strategy to integrate the traditional layered structure into an asymmetric but continuous porous network. Through infiltrations of microparticles and nanoparticles to targeted regions, active domains are created inside the porous scaffold versus having them applied externally. The fabricated internal active domains are highly adjustable in terms of its dimensions, pore size, and materials. We demonstrate that it is a general method that can be applicable to a wide variety of particles regardless of their material, dimensions, or geometry. By eliminating the external layered structure, problems such as those mentioned above can be eliminated. This integration technique can be extended to other devices required a layered structure, such as solid oxide fuel cells and lithium ion battery. PMID:24646923

  2. Porous Nanocomposites with Integrated Internal Domains: Application to Separation Membranes

    NASA Astrophysics Data System (ADS)

    Li, Wenle; Walz, John Y.

    2014-03-01

    Asymmetric membranes with layered structure have made significant achievements due to their balanced properties and multi-functionalities that come from a combination of multiple layers. However, issues such as delamination and substructure resistance are generated by the intrinsic layered structure. Here, we present a strategy to integrate the traditional layered structure into an asymmetric but continuous porous network. Through infiltrations of microparticles and nanoparticles to targeted regions, active domains are created inside the porous scaffold versus having them applied externally. The fabricated internal active domains are highly adjustable in terms of its dimensions, pore size, and materials. We demonstrate that it is a general method that can be applicable to a wide variety of particles regardless of their material, dimensions, or geometry. By eliminating the external layered structure, problems such as those mentioned above can be eliminated. This integration technique can be extended to other devices required a layered structure, such as solid oxide fuel cells and lithium ion battery.

  3. Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein

    PubMed Central

    Meacock, Suzanna L.; Lecomte, Fabienne J.L.; Crawshaw, Samuel G.; High, Stephen

    2002-01-01

    We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the α and β subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61α and Sec61β during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the “stage” of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide. PMID:12475939

  4. A Novel Family of Soluble Minimal Scaffolds Provides Structural Insight into the Catalytic Domains of Integral Membrane Metallopeptidases*

    PubMed Central

    López-Pelegrín, Mar; Cerdà-Costa, Núria; Martínez-Jiménez, Francisco; Cintas-Pedrola, Anna; Canals, Albert; Peinado, Juan R.; Marti-Renom, Marc A.; López-Otín, Carlos; Arolas, Joan L.; Gomis-Rüth, F. Xavier

    2013-01-01

    In the search for structural models of integral-membrane metallopeptidases (MPs), we discovered three related proteins from thermophilic prokaryotes, which we grouped into a novel family called “minigluzincins.” We determined the crystal structures of the zymogens of two of these (Pyrococcus abyssi proabylysin and Methanocaldococcus jannaschii projannalysin), which are soluble and, with ∼100 residues, constitute the shortest structurally characterized MPs to date. Despite relevant sequence and structural similarity, the structures revealed two unique mechanisms of latency maintenance through the C-terminal segments previously unseen in MPs as follows: intramolecular, through an extended tail, in proabylysin, and crosswise intermolecular, through a helix swap, in projannalysin. In addition, structural and sequence comparisons revealed large similarity with MPs of the gluzincin tribe such as thermolysin, leukotriene A4 hydrolase relatives, and cowrins. Noteworthy, gluzincins mostly contain a glutamate as third characteristic zinc ligand, whereas minigluzincins have a histidine. Sequence and structural similarity further allowed us to ascertain that minigluzincins are very similar to the catalytic domains of integral membrane MPs of the MEROPS database families M48 and M56, such as FACE1, HtpX, Oma1, and BlaR1/MecR1, which are provided with trans-membrane helices flanking or inserted into a minigluzincin-like catalytic domain. In a time where structural biochemistry of integral-membrane proteins in general still faces formidable challenges, the minigluzincin soluble minimal scaffold may contribute to our understanding of the working mechanisms of these membrane MPs and to the design of novel inhibitors through structure-aided rational drug design approaches. PMID:23733187

  5. The cytoplasmic domain is essential for transport function of the integral membrane transport protein SLC4A11.

    PubMed

    Loganathan, Sampath K; Lukowski, Chris M; Casey, Joseph R

    2016-01-15

    Large cytoplasmic domains (CD) are a common feature among integral membrane proteins. In virtually all cases, these CD have a function (e.g., binding cytoskeleton or regulatory factors) separate from that of the membrane domain (MD). Strong associations between CD and MD are rare. Here we studied SLC4A11, a membrane transport protein of corneal endothelial cells, the mutations of which cause genetic corneal blindness. SLC4A11 has a 41-kDa CD and a 57-kDa integral MD. One disease-causing mutation in the CD, R125H, manifests a catalytic defect, suggesting a role of the CD in transport function. Expressed in HEK-293 cells without the CD, MD-SLC4A11 is retained in the endoplasmic reticulum, indicating a folding defect. Replacement of CD-SLC4A11 with green fluorescent protein did not rescue MD-SLC4A11, suggesting some specific role of CD-SLC4A11. Homology modeling revealed that the structure of CD-SLC4A11 is similar to that of the Cl(-)/HCO3(-) exchange protein AE1 (SLC4A1) CD. Fusion to CD-AE1 partially rescued MD-SLC4A11 to the cell surface, suggesting that the structure of CD-AE1 is similar to that of CD-SLC4A11. The CD-AE1-MD-SLC4a11 chimera, however, had no functional activity. We conclude that CD-SLC4A11 has an indispensable role in the transport function of SLC4A11. CD-SLC4A11 forms insoluble precipitates when expressed in bacteria, suggesting that the domain cannot fold properly when expressed alone. Consistent with a strong association between CD-SLC4A11 and MD-SLC4A11, these domains specifically associate when coexpressed in HEK-293 cells. We conclude that SLC4A11 is a rare integral membrane protein in which the CD has strong associations with the integral MD, which contributes to membrane transport function. PMID:26582474

  6. Intracellular distribution of an integral nuclear pore membrane protein fused to green fluorescent protein--localization of a targeting domain.

    PubMed

    Söderqvist, H; Imreh, G; Kihlmark, M; Linnman, C; Ringertz, N; Hallberg, E

    1997-12-15

    The 121-kDa pore membrane protein (POM121) is a bitopic integral membrane protein specifically located in the pore membrane domain of the nuclear envelope with its short N-terminal tail exposed on the luminal side and its major C-terminal portion adjoining the nuclear pore complex. In order to locate a signal for targeting of POM121 to the nuclear pores, we overexpressed selected regions of POM121 alone or fused to the green fluorescent protein (GFP) in transiently transfected COS-1 cells or in a stably transfected neuroblastoma cell line. Microscopic analysis of the GFP fluorescence or immunostaining was used to determine the intracellular distribution of the overexpressed proteins. The endofluorescent GFP tag had no effect on the distribution of POM121, since the chimerical POM121-GFP fusion protein was correctly targeted to the nuclear pores of both COS-1 cells and neuroblastoma cells. Based on the differentiated intracellular sorting of the POM121 variants, we conclude that the first 128 amino acids of POM121 contains signals for targeting to the continuous endoplasmic reticulum/nuclear envelope membrane system but not specifically to the nuclear pores and that a specific nuclear pore targeting signal is located between amino acids 129 and 618 in the endoplasmically exposed portion of POM121. PMID:9461306

  7. P13, an Integral Membrane Protein of Borrelia burgdorferi, Is C-Terminally Processed and Contains Surface-Exposed Domains

    PubMed Central

    Noppa, Laila; Östberg, Yngve; Lavrinovicha, Marija; Bergström, Sven

    2001-01-01

    To elucidate antigens present on the bacterial surface of Borrelia burgdorferi sensu lato that may be involved in pathogenesis, we characterized a protein, P13, with an apparent molecular mass of 13 kDa. The protein was immunogenic and was expressed in large amounts during in vitro cultivation compared to other known antigens. An immunofluorescence assay, immunoelectron microscopy, and protease sensitivity assays indicated that P13 is surface exposed. The deduced sequence of the P13 peptide revealed a possible signal peptidase type I cleavage site, and computer analysis predicted that P13 is an integral membrane protein with three transmembrane-spanning domains. Mass spectrometry, in vitro translation, and N- and C-terminal amino acid sequencing analyses indicated that P13 was posttranslationally processed at both ends and modified by an unknown mechanism. Furthermore, p13 belongs to a gene family with five additional members in B. burgdorferi sensu stricto. The p13 gene is located on the linear chromosome of the bacterium, in contrast to five paralogous genes, which are located on extrachromosomal plasmids. The size of the p13 transcript was consistent with a monocistronic transcript. This new gene family may be involved in functions that are specific for this spirochete and its pathogenesis. PMID:11292755

  8. Characterization of lipid domains in erythrocyte membranes.

    PubMed Central

    Rodgers, W; Glaser, M

    1991-01-01

    Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species. Images PMID:1996337

  9. Membrane Domain Formation on Nanostructured Scaffolds

    NASA Astrophysics Data System (ADS)

    Collier, Charles; Liu, Fangjie; Srijanto, Bernadeta

    The spatial organization of lipids and proteins in biological membranes seems to have a functional role in the life of a cell. Separation of the lipids into distinct domains of greater order and anchoring to the cytoskeleton are two main mechanisms for organizing the membrane in cells. We propose a novel model membrane consisting of a lipid bilayer suspended over a nanostructured scaffold consisting of arrays of fabricated nanopillars. Unlike traditional model membranes, our model will have well-defined lateral structure and distributed substrate attachments that will emulate the connections of cellular membranes to the underlying cytoskeleton. Membranes will be characterized using neutron reflectometry, atomic force microscopy and fluorescence to verify a suspended, planar geometry with restricted diffusion at suspension points, and free diffusion in between. This architecture will allow the controlled study of lipid domain reorganization, viral infection and signal transduction that depend on the lateral structure of the membrane.

  10. Expression and characterization of the integral membrane domain of bacterial histidine kinase SCO3062 for structural studies

    SciTech Connect

    Yeo, Kwon Joo; Kwak, Su-Nam; Kim, Hyun Jung; Cheong, Chaejoon; Kim, Myung Hee; Jeon, Young Ho

    2008-11-14

    Bacterial histidine kinases play an important role in the response to external stimuli. Structural studies of the histidine kinase transmembrane domain are challenging due to difficulties in protein expression and sample preparation. After carrying out expression screening of a series of histidine kinases, we investigated sample preparation methods for obtaining high quality samples of the periplasmic and transmembrane domain (PTD) of the bacterial histidine kinase SCO3062. Various sample conditions were tested for their ability to give homogeneous NMR spectra of the SCO3062 PTD with well-resolved resonances. Circular dichroism and 3D {sup 15}N-edited NOESY spectrum results demonstrate that the SCO3062 PTD is predominantly {alpha}-helical. This method should be applicable to the NMR analysis of other transmembrane proteins.

  11. Matrix membranes and integrability

    SciTech Connect

    Zachos, C.; Fairlie, D.; Curtright, T.

    1997-06-01

    This is a pedagogical digest of results reported in Curtright, Fairlie, {ampersand} Zachos 1997, and an explicit implementation of Euler`s construction for the solution of the Poisson Bracket dual Nahm equation. But it does not cover 9 and 10-dimensional systems, and subsequent progress on them Fairlie 1997. Cubic interactions are considered in 3 and 7 space dimensions, respectively, for bosonic membranes in Poisson Bracket form. Their symmetries and vacuum configurations are explored. Their associated first order equations are transformed to Nahm`s equations, and are hence seen to be integrable, for the 3-dimensional case, by virtue of the explicit Lax pair provided. Most constructions introduced also apply to matrix commutator or Moyal Bracket analogs.

  12. The integrity of the alpha-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes.

    PubMed

    Franchini, G R; Storch, J; Corsico, B

    2008-04-01

    Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the alphaI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the alphaI-helix of LFABP (alpha(I)LbetaIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from alpha(I)LbetaIFABP compared to IFABP. The results indicate that the alphaI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the alphaII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the alphaI helix of IFABP in its physical interaction with membranes. PMID:18284926

  13. Attractive membrane domains control lateral diffusion.

    PubMed

    Forstner, Martin B; Martin, Douglas S; Rückerl, Florian; Käs, Josef A; Selle, Carsten

    2008-05-01

    Lipid membranes play a fundamental role in vital cellular functions such as signal transduction. Many of these processes rely on lateral diffusion within the membrane, generally a complex fluid containing ordered microdomains. However, little attention has been paid to the alterations in transport dynamics of a diffusing species caused by long-range interactions with membrane domains. In this paper, we address the effect of such interactions on diffusive transport by studying lateral diffusion in a phase-separated Langmuir phospholipid monolayer via single-particle tracking. We find that attractive dipole-dipole interactions between condensed phase domains and diffusing probe beads lead to transient confinement at the phase boundaries, causing a transition from two- to one-dimensional diffusion. Using Brownian dynamics simulations, the long-term diffusion constant for such a system is found to have a sensitive, Boltzmann-like, dependence on the interaction strength. In addition, this interaction strength is shown to be a strong function of the ratio of domain to particle size. As similar interactions are expected in biological membranes, the modulation of diffusive transport dynamics by varying interaction strength and/or domain size may offer cells selective spatial and temporal control over signaling processes. PMID:18643101

  14. Disrupting membrane raft domains by alkylphospholipids.

    PubMed

    Gomide, A B; Thomé, C H; dos Santos, G A; Ferreira, G A; Faça, V M; Rego, E M; Greene, L J; Stabeli, R G; Ciancaglini, P; Itri, R

    2013-05-01

    Using phase contrast and fluorescence microscopy we study the influence of the alkylphospholipid, ALP, 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate, ODPC, in giant unilamellar vesicles, GUVs, composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), brain sphingomyelin (SM) and cholesterol (Chol). The results show that adding 100μM ODPC (below CMC) to the outer solution of GUVs promotes DOPC membrane disruption over a period of 1h of continuous observation. On the other hand, the presence of SM and Chol in homogeneous fluid lipid bilayers protects the membrane from disruption. Interestingly, by adding 100μM ODPC to GUVs containing DOPC:SM:Chol (1:1:1), which display liquid ordered (Lo)-liquid disordered (Ld) phase coexistence, the domains rapidly disappear in less than 1min of ODPC contact with the membrane. The lipids are subsequently redistributed to liquid domains within a time course of 14-18min, reflecting that the homogenous phase was not thermodynamically stable, followed by rupture of the GUVs. A similar mechanism of action is also observed for perifosine, although to a larger extent. Therefore, the initial stage of lipid raft disruption by both ODPC and perifosine, and maybe other ALPS, by promoting lipid mixing, may be correlated with their toxicity upon neoplastic cells, since selective (dis)association of essential proteins within lipid raft microdomains must take place in the plasma membrane. PMID:23376656

  15. Composite membrane with integral rim

    SciTech Connect

    Routkevitch, Dmitri; Polyakov, Oleg G

    2015-01-27

    Composite membranes that are adapted for separation, purification, filtration, analysis, reaction and sensing. The composite membranes can include a porous support structure having elongate pore channels extending through the support structure. The composite membrane also includes an active layer comprising an active layer material, where the active layer material is completely disposed within the pore channels between the surfaces of the support structure. The active layer is intimately integrated within the support structure, thus enabling great robustness, reliability, resistance to mechanical stress and thermal cycling, and high selectivity. Methods for the fabrication of composite membranes are also provided.

  16. Lipid membrane domains in cell surface and vacuolar systems.

    PubMed

    Kobayashi, T; Hirabayashi, Y

    2000-01-01

    Detergent insoluble sphingolipid-cholesterol enriched 'raft'-like membrane microdomains have been implicated in a variety of biological processes including sorting, trafficking, and signaling. Mutant cells and knockout animals of sphingolipid biosynthesis are clearly useful to understand the biological roles of lipid components in raft-like domains. It is suggested that raft-like domains distribute in internal vacuolar membranes as well as plasma membranes. In addition to sphingolipid-cholesterol-rich membrane domains, recent studies suggest the existence of another lipid-membrane domain in the endocytic pathway. This domain is enriched with a unique phospholipid, lysobisphosphatidic acid (LBPA) and localized in the internal membrane of multivesicular endosome. LBPA-rich membrane domains are involved in lipid and protein sorting within the endosomal system. Possible interaction between sphingolipids and LBPA in sphingolipid-storage disease is discussed. PMID:11201787

  17. EH domain proteins regulate cardiac membrane protein targeting

    PubMed Central

    Gudmundsson, Hjalti; Hund, Thomas J.; Wright, Patrick J.; Kline, Crystal F.; Snyder, Jedidiah S.; Qian, Lan; Koval, Olha M.; Cunha, Shane R.; George, Manju; Rainey, Mark A.; Kashef, Farshid E.; Dun, Wen; Boyden, Penelope A.; Anderson, Mark E.; Band, Hamid; Mohler, Peter J.

    2010-01-01

    Rationale Cardiac membrane excitability is tightly regulated by an integrated network of membrane-associated ion channels, transporters, receptors, and signaling molecules. Membrane protein dynamics in health and disease are maintained by a complex ensemble of intracellular targeting, scaffolding, recycling, and degradation pathways. Surprisingly, despite decades of research linking dysfunction in membrane protein trafficking with human cardiovascular disease, essentially nothing is known regarding the molecular identity or function of these intracellular targeting pathways in excitable cardiomyocytes. Objective We sought to discover novel pathways for membrane protein targeting in primary cardiomyocytes. Methods and Results We report the initial characterization of a large family of membrane trafficking proteins in human heart. We employed a tissue-wide screen for novel ankyrin-associated trafficking proteins and identified four members of a unique Eps15 homology (EH) domain-containing protein family (EHD1, EHD2, EHD3, EHD4) that serve critical roles in endosome-based membrane protein targeting in other cell types. We show that EHD1-4 directly associate with ankyrin, provide the first information on the expression and localization of these molecules in primary cardiomyocytes, and demonstrate that EHD1-4 are co-expressed with ankyrin-B in the myocyte perinuclear region. Notably, the expression of multiple EHD proteins is increased in animal models lacking ankyrin-B, and EHD3-deficient cardiomyocytes display aberrant ankyrin-B localization and selective loss of Na/Ca exchanger expression and function. Finally, we report significant modulation of EHD expression following myocardial infarction, suggesting that these proteins may play a key role in regulating membrane excitability in normal and diseased heart. Conclusions Our findings identify and characterize a new class of cardiac trafficking proteins, define the first group of proteins associated with the ankyrin

  18. Effects of the oncogenic V(664)E mutation on membrane insertion, structure, and sequence-dependent interactions of the Neu transmembrane domain in micelles and model membranes: an integrated biophysical and simulation study.

    PubMed

    Beevers, Andrew J; Nash, Anthony; Salazar-Cancino, Martha; Scott, David J; Notman, Rebecca; Dixon, Ann M

    2012-03-27

    Receptor tyrosine kinases bind ligands such as cytokines, hormones, and growth factors and regulate key cellular processes, including cell division. They are also implicated in the development of many types of cancer. One such example is the Neu receptor tyrosine kinase found in rats (homologous to the human ErbB2 protein), which can undergo a valine to glutamic acid (V(664)E) mutation at the center of its α-helical transmembrane domain. This substitution results in receptor activation and oncogenesis. The molecular basis of this dramatic change in behavior upon introduction of the V(664)E mutation has been difficult to pin down, with conflicting results reported in the literature. Here we report the first quantitative, thermodynamic analysis of dimerization and biophysical characterization of the rat Neu transmembrane domain and several mutants in a range of chemical environments. These data have allowed us to identify the effects of the V(664)E mutation in the isolated TM domain with respect to protein-protein and protein-lipid interactions, membrane insertion, and secondary structure. We also report the results from a 100 ns atomistic molecular dynamics simulation of the Neu transmembrane domain in a model membrane bilayer (dipalmitoylphosphatidylcholine). The results from simulation and experiment are in close agreement and suggest that, in the model systems investigated, the V(664)E mutation leads to a weakening of the TM dimer and a change in sequence-dependent interactions. These results are contrary to recent results obtained in mammalian membranes, and the implications of this are discussed. PMID:22385253

  19. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    PubMed Central

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-01

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes. PMID:25635869

  20. Hydrophobic mismatch sorts SNARE proteins into distinct membrane domains

    NASA Astrophysics Data System (ADS)

    Milovanovic, Dragomir; Honigmann, Alf; Koike, Seiichi; Göttfert, Fabian; Pähler, Gesa; Junius, Meike; Müllar, Stefan; Diederichsen, Ulf; Janshoff, Andreas; Grubmüller, Helmut; Risselada, Herre J.; Eggeling, Christian; Hell, Stefan W.; van den Bogaart, Geert; Jahn, Reinhard

    2015-01-01

    The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein-protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes.

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

    PubMed Central

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

    2010-01-01

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

  2. Transient domain formation in membrane-bound organelles undergoing maturation

    NASA Astrophysics Data System (ADS)

    Dmitrieff, Serge; Sens, Pierre

    2013-12-01

    The membrane components of cellular organelles have been shown to segregate into domains as the result of biochemical maturation. We propose that the dynamical competition between maturation and lateral segregation of membrane components regulates domain formation. We study a two-component fluid membrane in which enzymatic reaction irreversibly converts one component into another and phase separation triggers the formation of transient membrane domains. The maximum domain size is shown to depend on the maturation rate as a power law similar to the one observed for domain growth with time in the absence of maturation, despite this time dependence not being verified in the case of irreversible maturation. This control of domain size by enzymatic activity could play a critical role in regulating exchange between organelles or within compartmentalized organelles such as the Golgi apparatus.

  3. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily

    PubMed Central

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  4. Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.

    PubMed

    Lenoir, Marc; Kufareva, Irina; Abagyan, Ruben; Overduin, Michael

    2015-01-01

    The human genome encodes about 285 proteins that contain at least one annotated pleckstrin homology (PH) domain. As the first phosphoinositide binding module domain to be discovered, the PH domain recruits diverse protein architectures to cellular membranes. PH domains constitute one of the largest protein superfamilies, and have diverged to regulate many different signaling proteins and modules such as Dbl homology (DH) and Tec homology (TH) domains. The ligands of approximately 70 PH domains have been validated by binding assays and complexed structures, allowing meaningful extrapolation across the entire superfamily. Here the Membrane Optimal Docking Area (MODA) program is used at a genome-wide level to identify all membrane docking PH structures and map their lipid-binding determinants. In addition to the linear sequence motifs which are employed for phosphoinositide recognition, the three dimensional structural features that allow peripheral membrane domains to approach and insert into the bilayer are pinpointed and can be predicted ab initio. The analysis shows that conserved structural surfaces distinguish which PH domains associate with membrane from those that do not. Moreover, the results indicate that lipid-binding PH domains can be classified into different functional subgroups based on the type of membrane insertion elements they project towards the bilayer. PMID:26512702

  5. Membrane-Sculpting BAR Domains Generate Stable Lipid Microdomains

    PubMed Central

    Zhao, Hongxia; Michelot, Alphée; Koskela, Essi V.; Tkach, Vadym; Stamou, Dimitrios; Drubin, David G.; Lappalainen, Pekka

    2014-01-01

    SUMMARY Bin-Amphiphysin-Rvs (BAR) domain proteins are central regulators of many cellular processes involving membrane dynamics. BAR domains sculpt phosphoinositide-rich membranes to generate membrane protrusions or invaginations. Here, we report that, in addition to regulating membrane geometry, BAR domains can generate extremely stable lipid microdomains by “freezing” phosphoinositide dynamics. This is a general feature of BAR domains, because the yeast endocytic BAR and Fes/CIP4 homology BAR (F-BAR) domains, the inverse BAR domain of Pinkbar, and the eisosomal BAR protein Lsp1 induced phosphoinositide clustering and halted lipid diffusion, despite differences in mechanisms of membrane interactions. Lsp1 displays comparable low diffusion rates in vitro and in vivo, suggesting that BAR domain proteins also generate stable phosphoinositide microdomains in cells. These results uncover a conserved role for BAR superfamily proteins in regulating lipid dynamics within membranes. Stable microdomains induced by BAR domain scaffolds and specific lipids can generate phase boundaries and diffusion barriers, which may have profound impacts on diverse cellular processes. PMID:24055060

  6. Superdiffusive motion of membrane-targeting C2 domains

    PubMed Central

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

    2015-01-01

    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. PMID:26639944

  7. Superdiffusive motion of membrane-targeting C2 domains

    NASA Astrophysics Data System (ADS)

    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.

  8. Membrane shape instabilities induced by BAR domain proteins

    NASA Astrophysics Data System (ADS)

    Baumgart, Tobias

    2014-03-01

    Membrane curvature has developed into a forefront of membrane biophysics. Numerous proteins involved in membrane curvature sensing and membrane curvature generation have recently been discovered, including proteins containing the crescent-shaped BAR domain as membrane binding and shaping module. Accordingly, the structure determination of these proteins and their multimeric complexes is increasingly well-understood. Substantially less understood, however, are thermodynamic and kinetic aspects and the detailed mechanisms of how these proteins interact with membranes in a curvature-dependent manner. New experimental approaches need to be combined with established techniques to be able to fill in these missing details. Here we use model membrane systems in combination with a variety of biophysical techniques to characterize mechanistic aspects of BAR domain protein function. This includes a characterization of membrane curvature sensing and membrane generation. We also establish kinetic and thermodynamic aspects of BAR protein dimerization in solution, and investigate kinetic aspects of membrane binding. We present two new approaches to investigate membrane shape instabilities and demonstrate that membrane shape instabilities can be controlled by protein binding and lateral membrane tension. This work is supported through NIH grant GM-097552 and NSF grant CBET-1053857.

  9. Calmodulin Promotes N-BAR Domain-Mediated Membrane Constriction and Endocytosis.

    PubMed

    Myers, Margaret D; Ryazantsev, Sergey; Hicke, Linda; Payne, Gregory S

    2016-04-18

    Membrane remodeling by BAR (Bin, Amphiphysin, RVS) domain-containing proteins, such as endophilins and amphiphysins, is integral to the process of endocytosis. However, little is known about the regulation of endocytic BAR domain activity. We have identified an interaction between the yeast Rvs167 N-BAR domain and calmodulin. Calmodulin-binding mutants of Rvs167 exhibited defects in endocytic vesicle release. In vitro, calmodulin enhanced membrane tubulation and constriction by wild-type Rvs167 but not calmodulin-binding-defective mutants. A subset of mammalian N-BAR domains bound calmodulin, and co-expression of calmodulin with endophilin A2 potentiated tubulation in vivo. These studies reveal a conserved role for calmodulin in regulating the intrinsic membrane-sculpting activity of endocytic N-BAR domains. PMID:27093085

  10. Separation and characterization of late endosomal membrane domains.

    PubMed

    Kobayashi, Toshihide; Beuchat, Marie-Hélène; Chevallier, Julien; Makino, Asami; Mayran, Nathalie; Escola, Jean-Michel; Lebrand, Cecile; Cosson, Pierre; Kobayashi, Tetsuyuki; Gruenberg, Jean

    2002-08-30

    Very little is known about the biophysical properties and the lipid or protein composition of membrane domains presumably present in endocytic and biosynthetic organelles. Here we analyzed the membrane composition of late endosomes by suborganellar fractionation in the absence of detergent. We found that the internal membranes of this multivesicular organelle can be separated from the limiting membrane and that each membrane population exhibited a defined composition. Our data also indicated that internal membranes may consist of at least two populations, containing primarily phosphatidylcholine or lysobisphosphatidic acid as major phospholipid, arguing for the existence of significant microheterogeneity within late endosomal membranes. We also found that lysobisphosphatidic acid exhibited unique pH-dependent fusogenic properties, and we speculated that this lipid is an ideal candidate to regulate the dynamic properties of this internal membrane mosaic. PMID:12065580

  11. SH4-domain-induced plasma membrane dynamization promotes bleb-associated cell motility.

    PubMed

    Tournaviti, Stella; Hannemann, Sebastian; Terjung, Stefan; Kitzing, Thomas M; Stegmayer, Carolin; Ritzerfeld, Julia; Walther, Paul; Grosse, Robert; Nickel, Walter; Fackler, Oliver T

    2007-11-01

    SH4 domains provide bipartite membrane-targeting signals for oncogenic Src family kinases. Here we report the induction of non-apoptotic plasma membrane (PM) blebbing as a novel and conserved activity of SH4 domains derived from the prototypic Src kinases Src, Fyn, Yes and Lck as well as the HASPB protein of Leishmania parasites. SH4-domain-induced blebbing is highly dynamic, with bleb formation and collapse displaying distinct kinetics. These reorganizations of the PM are controlled by Rho but not Rac or Cdc42 GTPase signalling pathways. SH4-induced membrane blebbing requires the membrane association of the SH4 domain, is regulated by the activities of Rock kinase and myosin II ATPase, and depends on the integrity of F-actin as well as microtubules. Endogenous Src kinase activity is crucial for PM blebbing in SH4-domain-expressing cells, active Src and Rock kinases are enriched in SH4-domain-induced PM blebs, and PM blebbing correlates with enhanced cell invasion in 3D matrices. These results establish a novel link between SH4 domains, Src activity and Rho signalling, and implicate SH4-domain-mediated PM dynamization as a mechanism that influences invasiveness of cells transformed by SH4-domain-containing oncoproteins. PMID:17959630

  12. A PH domain in ACAP1 possesses key features of the BAR domain in promoting membrane curvature.

    PubMed

    Pang, Xiaoyun; Fan, Jun; Zhang, Yan; Zhang, Kai; Gao, Bingquan; Ma, Jun; Li, Jian; Deng, Yuchen; Zhou, Qiangjun; Egelman, Edward H; Hsu, Victor W; Sun, Fei

    2014-10-13

    The BAR (Bin-Amphiphysin-Rvs) domain undergoes dimerization to produce a curved protein structure, which superimposes onto membrane through electrostatic interactions to sense and impart membrane curvature. In some cases, a BAR domain also possesses an amphipathic helix that inserts into the membrane to induce curvature. ACAP1 (Arfgap with Coil coil, Ankyrin repeat, and PH domain protein 1) contains a BAR domain. Here, we show that this BAR domain can neither bind membrane nor impart curvature, but instead requires a neighboring PH (Pleckstrin Homology) domain to achieve these functions. Specific residues within the PH domain are responsible for both membrane binding and curvature generation. The BAR domain adjacent to the PH domain instead interacts with the BAR domains of neighboring ACAP1 proteins to enable clustering at the membrane. Thus, we have uncovered the molecular basis for an unexpected and unconventional collaboration between PH and BAR domains in membrane bending. PMID:25284369

  13. The Observation of Highly Ordered Domains in Membranes with Cholesterol

    SciTech Connect

    Armstrong, Clare L; Marquardt, Drew; Dies, Hannah; Kucerka, Norbert; Yamani, Zahra; Harroun, Thad; Katsaras, John; Shi, A-C; Rheinstadter, Maikel C

    2013-01-01

    Rafts, or functional domains, are transient nano- or mesoscopic structures in the exoplasmic leaflet of the plasma membrane, and are thought to be essential for many cellular processes. Using neutron diffraction and computer modelling, we present evidence for the existence of highly ordered lipid domains in the cholesterol-rich (32.5 mol%) liquid-ordered (lo) phase of dipalmitoylphosphatidylcholine membranes. The liquid ordered phase in one-component lipid membranes has previously been thought to be a homogeneous phase. The presence of highly ordered lipid domains embedded in a disordered lipid matrix implies non-uniform distribution of cholesterol between the two phases. The experimental results are in excellent agreement with recent computer simulations of DPPC/cholesterol complexes [Meinhardt, Vink and Schmid (2013). Proc Natl Acad Sci USA 110(12): 4476 4481], which reported the existence of nanometer size lo domains in a liquid disordered lipid environment.

  14. The Observation of Highly Ordered Domains in Membranes with Cholesterol

    PubMed Central

    Armstrong, Clare L.; Marquardt, Drew; Dies, Hannah; Kučerka, Norbert; Yamani, Zahra; Harroun, Thad A.; Katsaras, John; Shi, An-Chang; Rheinstädter, Maikel C.

    2013-01-01

    Rafts, or functional domains, are transient nano- or mesoscopic structures in the exoplasmic leaflet of the plasma membrane, and are thought to be essential for many cellular processes. Using neutron diffraction and computer modelling, we present evidence for the existence of highly ordered lipid domains in the cholesterol-rich (32.5 mol%) liquid-ordered () phase of dipalmitoylphosphatidylcholine membranes. The liquid ordered phase in one-component lipid membranes has previously been thought to be a homogeneous phase. The presence of highly ordered lipid domains embedded in a disordered lipid matrix implies non-uniform distribution of cholesterol between the two phases. The experimental results are in excellent agreement with recent computer simulations of DPPC/cholesterol complexes [Meinhardt, Vink and Schmid (2013). Proc Natl Acad Sci USA 110(12): 4476–4481], which reported the existence of nanometer size domains in a liquid disordered lipid environment. PMID:23823623

  15. Random pinning limits the size of membrane adhesion domains

    NASA Astrophysics Data System (ADS)

    Speck, Thomas; Vink, Richard L. C.

    2012-09-01

    Theoretical models describing specific adhesion of membranes predict (for certain parameters) a macroscopic phase separation of bonds into adhesion domains. We show that this behavior is fundamentally altered if the membrane is pinned randomly due to, e.g., proteins that anchor the membrane to the cytoskeleton. Perturbations which locally restrict membrane height fluctuations induce quenched disorder of the random-field type. This rigorously prevents the formation of macroscopic adhesion domains following the Imry-Ma argument [Imry and Ma, Phys. Rev. Lett.10.1103/PhysRevLett.35.1399 35, 1399 (1975)]. Our prediction of random-field disorder follows from analytical calculations and is strikingly confirmed in large-scale Monte Carlo simulations. These simulations are based on an efficient composite Monte Carlo move, whereby membrane height and bond degrees of freedom are updated simultaneously in a single move. The application of this move should prove rewarding for other systems also.

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

    PubMed Central

    Wang, Shen; Li, Yun; Ma, Cong

    2016-01-01

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

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

    PubMed

    Wang, Shen; Li, Yun; Ma, Cong

    2016-01-01

    Synaptotagmin-1 (Syt1) acts as a Ca(2+) sensor for neurotransmitter release through its C2 domains. It has been proposed that Syt1 promotes SNARE-dependent fusion mainly through its C2B domain, but the underlying mechanism is poorly understood. In this study, we show that the C2B domain interacts simultaneously with acidic membranes and SNARE complexes via the top Ca(2+)-binding loops, the side polybasic patch, and the bottom face in response to Ca(2+). Disruption of the simultaneous interactions completely abrogates the triggering activity of the C2B domain in liposome fusion. We hypothesize that the simultaneous interactions endow the C2B domain with an ability to deform local membranes, and this membrane-deformation activity might underlie the functional significance of the Syt1 C2B domain in vivo. PMID:27083046

  18. Efficient integration method for fictitious domain approaches

    NASA Astrophysics Data System (ADS)

    Duczek, Sascha; Gabbert, Ulrich

    2015-10-01

    In the current article, we present an efficient and accurate numerical method for the integration of the system matrices in fictitious domain approaches such as the finite cell method (FCM). In the framework of the FCM, the physical domain is embedded in a geometrically larger domain of simple shape which is discretized using a regular Cartesian grid of cells. Therefore, a spacetree-based adaptive quadrature technique is normally deployed to resolve the geometry of the structure. Depending on the complexity of the structure under investigation this method accounts for most of the computational effort. To reduce the computational costs for computing the system matrices an efficient quadrature scheme based on the divergence theorem (Gauß-Ostrogradsky theorem) is proposed. Using this theorem the dimension of the integral is reduced by one, i.e. instead of solving the integral for the whole domain only its contour needs to be considered. In the current paper, we present the general principles of the integration method and its implementation. The results to several two-dimensional benchmark problems highlight its properties. The efficiency of the proposed method is compared to conventional spacetree-based integration techniques.

  19. Kinetics of Domains Registration in Multicomponent Lipid Bilayer Membranes

    PubMed Central

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

    2014-01-01

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

  20. Domain Organization of Membrane-Bound Factor VIII

    PubMed Central

    Stoilova-McPhie, Svetla; Lynch, Gillian C.; Ludtke, Steven; Pettitt, B. Montgomery

    2014-01-01

    Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the cofactor to the serine protease factor IXa (FIXa) within the membrane-bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal clot formation. FVIII is composed of two noncovalently linked peptide chains: a light chain (LC) holding the membrane interaction sites and a heavy chain (HC) holding the main FIXa interaction sites. The interplay between the light and heavy chains (HCs) in the membrane-bound state is critical for the biological efficiency of FVIII. Here, we present our cryo-electron microscopy (EM) and structure analysis studies of human FVIII-LC, when helically assembled onto negatively charged single lipid bilayer nanotubes. The resolved FVIII-LC membrane-bound structure supports aspects of our previously proposed FVIII structure from membrane-bound two-dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane. The LC is oriented differently in the FVIII membrane-bound helical and 2D crystal structures based on EM data, and the existing X-ray structures. This flexibility of the FVIII-LC domain organization in different states is discussed in the light of the FVIIIa-FIXa complex assembly and function. PMID:23616213

  1. VAMP-1: a synaptic vesicle-associated integral membrane protein.

    PubMed

    Trimble, W S; Cowan, D M; Scheller, R H

    1988-06-01

    Several proteins are associated with, or are integral components of, the lipid bilayer that forms the delineating membrane of neuronal synaptic vesicles. To characterize these molecules, we used a polyclonal antiserum raised against purified cholinergic synaptic vesicles from Torpedo to screen a cDNA expression library constructed from mRNA of the electromotor nucleus. One clone encodes VAMP-1 (vesicle-associated membrane protein 1), a nervous-system-specific protein of 120 amino acids whose primary sequence can be divided into three domains: a proline-rich amino terminus, a highly charged internal region, and a hydrophobic carboxyl-terminal domain that is predicted to comprise a membrane anchor. Tryptic digestion of intact and lysed vesicles suggests that the protein faces the cytoplasm, where it may play a role in packaging, transport, or release of neurotransmitters. PMID:3380805

  2. Kinetics of enzymatic reactions in lipid membranes containing domains

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.; Höök, Fredrik

    2015-04-01

    An appreciable part of enzymes operating in vivo is associated with lipid membranes. The function of such enzymes can be influenced by the presence of domains containing proteins and/or composed of different lipids. The corresponding experimental model-system studies can be performed under well controlled conditions, e.g., on a planar supported lipid bilayer or surface-immobilized vesicles. To clarify what may happen in such systems, we propose general kinetic equations describing the enzyme-catalyzed substrate conversion occurring via the Michaelis-Menten (MM) mechanism on a membrane with domains which do not directly participate in reaction. For two generic situations when a relatively slow reaction takes place primarily in or outside domains, we take substrate saturation and lateral substrate-substrate interactions at domains into account and scrutinize the dependence of the reaction rate on the average substrate coverage. With increasing coverage, depending on the details, the reaction rate reaches saturation via an inflection point or monotonously as in the conventional MM case. In addition, we show analytically the types of reaction kinetics occurring primarily at domain boundaries. In the physically interesting situation when the domain growth is fast on the reaction time scale, the latter kinetics are far from conventional. The opposite situation when the reaction is fast and controlled by diffusion has been studied by using the Monte Carlo technique. The corresponding results indicate that the dependence of the reaction kinetics on the domain size may be weak.

  3. Adaptive Lipid Packing and Bioactivity in Membrane Domains

    PubMed Central

    Sezgin, Erdinc; Gutmann, Theresia; Buhl, Tomasz; Dirkx, Ron; Grzybek, Michal; Coskun, Ünal; Solimena, Michele; Simons, Kai; Levental, Ilya; Schwille, Petra

    2015-01-01

    Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells. PMID:25905447

  4. Structure of the membrane domain of respiratory complex I.

    PubMed

    Efremov, Rouslan G; Sazanov, Leonid A

    2011-08-25

    Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0 Å resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements. PMID:21822288

  5. Designing lipids for selective partitioning into liquid ordered membrane domains.

    PubMed

    Momin, Noor; Lee, Stacey; Gadok, Avinash K; Busch, David J; Bachand, George D; Hayden, Carl C; Stachowiak, Jeanne C; Sasaki, Darryl Y

    2015-04-28

    Self-organization of lipid molecules into specific membrane phases is key to the development of hierarchical molecular assemblies that mimic cellular structures. While the packing interaction of the lipid tails should provide the major driving force to direct lipid partitioning to ordered or disordered membrane domains, numerous examples show that the headgroup and spacer play important but undefined roles. We report here the development of several new biotinylated lipids that examine the role of spacer chemistry and structure on membrane phase partitioning. The new lipids were prepared with varying lengths of low molecular weight polyethylene glycol (EGn) spacers to examine how spacer hydrophilicity and length influence their partitioning behavior following binding with FITC-labeled streptavidin in liquid ordered (Lo) and liquid disordered (Ld) phase coexisting membranes. Partitioning coefficients (Kp Lo/Ld) of the biotinylated lipids were determined using fluorescence measurements in studies with giant unilamellar vesicles (GUVs). Compared against DPPE-biotin, DPPE-cap-biotin, and DSPE-PEG2000-biotin lipids, the new dipalmityl-EGn-biotin lipids exhibited markedly enhanced partitioning into liquid ordered domains, achieving Kp of up to 7.3 with a decaethylene glycol spacer (DP-EG10-biotin). We further demonstrated biological relevance of the lipids with selective partitioning to lipid raft-like domains observed in giant plasma membrane vesicles (GPMVs) derived from mammalian cells. Our results found that the spacer group not only plays a pivotal role for designing lipids with phase selectivity but may also influence the structural order of the domain assemblies. PMID:25772372

  6. Spatially distinct and metabolically active membrane domain in mycobacteria.

    PubMed

    Hayashi, Jennifer M; Luo, Chu-Yuan; Mayfield, Jacob A; Hsu, Tsungda; Fukuda, Takeshi; Walfield, Andrew L; Giffen, Samantha R; Leszyk, John D; Baer, Christina E; Bennion, Owen T; Madduri, Ashoka; Shaffer, Scott A; Aldridge, Bree B; Sassetti, Christopher M; Sandler, Steven J; Kinoshita, Taroh; Moody, D Branch; Morita, Yasu S

    2016-05-10

    Protected from host immune attack and antibiotic penetration by their unique cell envelope, mycobacterial pathogens cause devastating human diseases such as tuberculosis. Seamless coordination of cell growth with cell envelope elongation at the pole maintains this barrier. Unraveling this spatiotemporal regulation is a potential strategy for controlling mycobacterial infections. Our biochemical analysis previously revealed two functionally distinct membrane fractions in Mycobacterium smegmatis cell lysates: plasma membrane tightly associated with the cell wall (PM-CW) and a distinct fraction of pure membrane free of cell wall components (PMf). To provide further insight into the functions of these membrane fractions, we took the approach of comparative proteomics and identified more than 300 proteins specifically associated with the PMf, including essential enzymes involved in cell envelope synthesis such as a mannosyltransferase, Ppm1, and a galactosyltransferase, GlfT2. Furthermore, comparative lipidomics revealed the distinct lipid composition of the PMf, with specific association of key cell envelope biosynthetic precursors. Live-imaging fluorescence microscopy visualized the PMf as patches of membrane spatially distinct from the PM-CW and notably enriched in the pole of the growing cells. Taken together, our study provides the basis for assigning the PMf as a spatiotemporally distinct and metabolically active membrane domain involved in cell envelope biogenesis. PMID:27114527

  7. Transmembrane helices can induce domain formation in crowded model membranes.

    PubMed

    Domański, Jan; Marrink, Siewert J; Schäfer, Lars V

    2012-04-01

    We studied compositionally heterogeneous multi-component model membranes comprised of saturated lipids, unsaturated lipids, cholesterol, and α-helical TM protein models using coarse-grained molecular dynamics simulations. Reducing the mismatch between the length of the saturated and unsaturated lipid tails reduced the driving force for segregation into liquid-ordered (l(o)) and liquid-disordered (l(d)) lipid domains. Cholesterol depletion had a similar effect, and binary lipid mixtures without cholesterol did not undergo large-scale phase separation under the simulation conditions. The phase-separating ternary dipalmitoyl-phosphatidylcholine (DPPC)/dilinoleoyl-PC (DLiPC)/cholesterol bilayer was found to segregate into l(o) and l(d) domains also in the presence of a high concentration of ΤΜ helices. The l(d) domain was highly crowded with TM helices (protein-to-lipid ratio ~1:5), slowing down lateral diffusion by a factor of 5-10 as compared to the dilute case, with anomalous (sub)-diffusion on the μs time scale. The membrane with the less strongly unsaturated palmitoyl-linoleoyl-PC instead of DLiPC, which in the absence of TM α-helices less strongly deviated from ideal mixing, could be brought closer to a miscibility critical point by introducing a high concentration of TM helices. Finally, the 7-TM protein bacteriorhodopsin was found to partition into the l(d) domains irrespective of hydrophobic matching. These results show that it is possible to directly study the lateral reorganization of lipids and proteins in compositionally heterogeneous and crowded model biomembranes with coarse-grained molecular dynamics simulations, a step toward simulations of realistic, compositionally complex cellular membranes. This article is part of a Special Issue entitled: Protein Folding in Membranes. PMID:21884678

  8. Domain formation in membranes caused by lipid wetting of protein.

    PubMed

    Akimov, Sergey A; Frolov, Vladimir A J; Kuzmin, Peter I; Zimmerberg, Joshua; Chizmadzhev, Yuri A; Cohen, Fredric S

    2008-05-01

    Formation of rafts and other domains in cell membranes is considered as wetting of proteins by lipids. The membrane is modeled as a continuous elastic medium. Thermodynamic functions of the lipid films that wet proteins are calculated using a mean-field theory of liquid crystals as adapted to biomembranes. This approach yields the conditions necessary for a macroscopic wetting film to form; its thickness could also be determined. It is shown that films of macroscopic thicknesses form around large (tens nanometers in diameter) lipid-protein aggregates; only thin adsorption films form around single proteins or small complexes. The means by which wetting films can facilitate the merger of these aggregates is considered. It is shown that a wetting film prevents a protein from leaving an aggregate. Using experimentally derived values of elastic moduli and spontaneous curvatures as well as height mismatch between aggregates and bulk membrane, we obtained numerical results, which can be compared with the experimental data. PMID:18643096

  9. Thermodynamics of Membrane Insertion and Refolding of the Diphtheria Toxin T-Domain.

    PubMed

    Vargas-Uribe, Mauricio; Rodnin, Mykola V; Öjemalm, Karin; Holgado, Aurora; Kyrychenko, Alexander; Nilsson, IngMarie; Posokhov, Yevgen O; Makhatadze, George; von Heijne, Gunnar; Ladokhin, Alexey S

    2015-06-01

    The diphtheria toxin translocation (T) domain inserts into the endosomal membrane in response to the endosomal acidification and enables the delivery of the catalytic domain into the cell. The insertion pathway consists of a series of conformational changes that occur in solution and in the membrane and leads to the conversion of a water-soluble state into a transmembrane state. In this work, we utilize various biophysical techniques to characterize the insertion pathway from the thermodynamic perspective. Thermal and chemical unfolding measured by differential scanning calorimetry, circular dichroism, and tryptophan fluorescence reveal that the free energy of unfolding of the T-domain at neutral and mildly acidic pH differ by 3-5 kcal/mol, depending on the experimental conditions. Fluorescence correlation spectroscopy measurements show that the free energy change from the membrane-competent state to the interfacial state is approximately -8 kcal/mol and is pH-independent, while that from the membrane-competent state to the transmembrane state ranges between -9.5 and -12 kcal/mol, depending on the membrane lipid composition and pH. Finally, the thermodynamics of transmembrane insertion of individual helices was tested using an in vitro assay that measures the translocon-assisted integration of test sequences into the microsomal membrane. These experiments suggest that even the most hydrophobic helix TH8 has only a small favorable free energy of insertion. The free energy for the insertion of the consensus insertion unit TH8-TH9 is slightly more favorable, yet less favorable than that measured for the entire protein, suggesting a cooperative effect for the membrane insertion of the helices of the T-domain. PMID:25281329

  10. Layilin, a Novel Integral Membrane Protein, Is a Hyaluronan Receptor

    PubMed Central

    Bono, Petri; Rubin, Kristofer; Higgins, Jonathan M. G.; Hynes, Richard O.

    2001-01-01

    The actin cytoskeleton plays a significant role in changes of cell shape and motility, and interactions between the actin filaments and the cell membrane are crucial for a variety of cellular processes. Several adaptor proteins, including talin, maintain the cytoskeleton-membrane linkage by binding to integral membrane proteins and to the cytoskeleton. Layilin, a recently characterized transmembrane protein with homology to C-type lectins, is a membrane-binding site for talin in peripheral ruffles of spreading cells. To facilitate studies of layilin's function, we have generated a layilin-Fc fusion protein comprising the extracellular part of layilin joined to human immunoglobulin G heavy chain and used this chimera to identify layilin ligands. Here, we demonstrate that layilin-Fc fusion protein binds to hyaluronan immobilized to Sepharose. Microtiter plate-binding assays, coprecipitation experiments, and staining of sections predigested with different glycosaminoglycan-degrading enzymes and cell adhesion assays all revealed that layilin binds specifically to hyaluronan but not to other tested glycosaminoglycans. Layilin's ability to bind hyaluronan, a ubiquitous extracellular matrix component, reveals an interesting parallel between layilin and CD44, because both can bind to cytoskeleton-membrane linker proteins through their cytoplasmic domains and to hyaluronan through their extracellular domains. This parallelism suggests a role for layilin in cell adhesion and motility. PMID:11294894

  11. Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family1[C][W

    PubMed Central

    Roppolo, Daniele; Boeckmann, Brigitte; Pfister, Alexandre; Boutet, Emmanuel; Rubio, Maria C.; Dénervaud-Tendon, Valérie; Vermeer, Joop E.M.; Gheyselinck, Jacqueline; Xenarios, Ioannis; Geldner, Niko

    2014-01-01

    CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells. PMID:24920445

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

    PubMed Central

    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

  13. Sterol-Rich Membrane Domains Define Fission Yeast Cell Polarity.

    PubMed

    Makushok, Tatyana; Alves, Paulo; Huisman, Stephen Michiel; Kijowski, Adam Rafal; Brunner, Damian

    2016-05-19

    Cell polarization is crucial for the functioning of all organisms. The cytoskeleton is central to the process but its role in symmetry breaking is poorly understood. We study cell polarization when fission yeast cells exit starvation. We show that the basis of polarity generation is de novo sterol biosynthesis, cell surface delivery of sterols, and their recruitment to the cell poles. This involves four phases occurring independent of the polarity factor cdc42p. Initially, multiple, randomly distributed sterol-rich membrane (SRM) domains form at the plasma membrane, independent of the cytoskeleton and cell growth. These domains provide platforms on which the growth and polarity machinery assembles. SRM domains are then polarized by the microtubule-dependent polarity factor tea1p, which prepares for monopolar growth initiation and later switching to bipolar growth. SRM polarization requires F-actin but not the F-actin organizing polarity factors for3p and bud6p. We conclude that SRMs are key to cell polarization. PMID:27180904

  14. Integrated Ceramic Membrane System for Hydrogen Production

    SciTech Connect

    Schwartz, Joseph; Lim, Hankwon; Drnevich, Raymond

    2010-08-05

    Phase I was a technoeconomic feasibility study that defined the process scheme for the integrated ceramic membrane system for hydrogen production and determined the plan for Phase II. The hydrogen production system is comprised of an oxygen transport membrane (OTM) and a hydrogen transport membrane (HTM). Two process options were evaluated: 1) Integrated OTM-HTM reactor – in this configuration, the HTM was a ceramic proton conductor operating at temperatures up to 900°C, and 2) Sequential OTM and HTM reactors – in this configuration, the HTM was assumed to be a Pd alloy operating at less than 600°C. The analysis suggested that there are no technical issues related to either system that cannot be managed. The process with the sequential reactors was found to be more efficient, less expensive, and more likely to be commercialized in a shorter time than the single reactor. Therefore, Phase II focused on the sequential reactor system, specifically, the second stage, or the HTM portion. Work on the OTM portion was conducted in a separate program. Phase IIA began in February 2003. Candidate substrate materials and alloys were identified and porous ceramic tubes were produced and coated with Pd. Much effort was made to develop porous substrates with reasonable pore sizes suitable for Pd alloy coating. The second generation of tubes showed some improvement in pore size control, but this was not enough to get a viable membrane. Further improvements were made to the porous ceramic tube manufacturing process. When a support tube was successfully coated, the membrane was tested to determine the hydrogen flux. The results from all these tests were used to update the technoeconomic analysis from Phase I to confirm that the sequential membrane reactor system can potentially be a low-cost hydrogen supply option when using an existing membrane on a larger scale. Phase IIB began in October 2004 and focused on demonstrating an integrated HTM/water gas shift (WGS) reactor to

  15. Dendritic Domains with Hexagonal Symmetry Formed by X-Shaped Bolapolyphiles in Lipid Membranes

    PubMed Central

    Werner, Stefan; Ebert, Helgard; Lechner, Bob-Dan; Lange, Frank; Achilles, Anja; Bärenwald, Ruth; Poppe, Silvio; Blume, Alfred; Saalwächter, Kay; Tschierske, Carsten; Bacia, Kirsten

    2015-01-01

    A novel class of bolapolyphile (BP) molecules are shown to integrate into phospholipid bilayers and self-assemble into unique sixfold symmetric domains of snowflake-like dendritic shapes. The BPs comprise three philicities: a lipophilic, rigid, π–π stacking core; two flexible lipophilic side chains; and two hydrophilic, hydrogen-bonding head groups. Confocal microscopy, differential scanning calorimetry, XRD, and solid-state NMR spectroscopy confirm BP-rich domains with transmembrane-oriented BPs and three to four lipid molecules per BP. Both species remain well organized even above the main 1,2-dipalmitoyl-sn-glycero-3-phosphocholine transition. The BP molecules only dissolve in the fluid membrane above 70 °C. Structural variations of the BP demonstrate that head-group hydrogen bonding is a prerequisite for domain formation. Independent of the head group, the BPs reduce membrane corrugation. In conclusion, the BPs form nanofilaments by π stacking of aromatic cores, which reduce membrane corrugation and possibly fuse into a hexagonal network in the dendritic domains. PMID:25940233

  16. Role of tetanus neurotoxin insensitive vesicle-associated membrane protein in membrane domains transport and homeostasis

    PubMed Central

    Molino, Diana; Nola, Sébastien; Lam, Sin Man; Verraes, Agathe; Proux-Gillardeaux, Véronique; Boncompain, Gaëlle; Perez, Franck; Wenk, Markus; Shui, Guanghou; Danglot, Lydia; Galli, Thierry

    2015-01-01

    Biological membranes in eukaryotes contain a large variety of proteins and lipids often distributed in domains in plasma membrane and endomembranes. Molecular mechanisms responsible for the transport and the organization of these membrane domains along the secretory pathway still remain elusive. Here we show that vesicular SNARE TI-VAMP/VAMP7 plays a major role in membrane domains composition and transport. We found that the transport of exogenous and endogenous GPI-anchored proteins was altered in fibroblasts isolated from VAMP7-knockout mice. Furthermore, disassembly and reformation of the Golgi apparatus induced by Brefeldin A treatment and washout were impaired in VAMP7-depleted cells, suggesting that loss of VAMP7 expression alters biochemical properties and dynamics of the Golgi apparatus. In addition, lipid profiles from these knockout cells indicated a defect in glycosphingolipids homeostasis. We conclude that VAMP7 is required for effective transport of GPI–anchored proteins to cell surface and that VAMP7-dependent transport contributes to both sphingolipids and Golgi homeostasis. PMID:26196023

  17. Formation of functional cell membrane domains: the interplay of lipid- and protein-mediated interactions.

    PubMed Central

    Harder, Thomas

    2003-01-01

    Numerous cell membrane associated processes, including signal transduction, membrane sorting, protein processing and virus trafficking take place in membrane subdomains. Protein-protein interactions provide the frameworks necessary to generate biologically functional membrane domains. For example, coat proteins define membrane areas destined for sorting processes, viral proteins self-assemble to generate a budding virus, and adapter molecules organize multimolecular signalling assemblies, which catalyse downstream reactions. The concept of raft lipid-based membrane domains provides a different principle for compartmentalization and segregation of membrane constituents. Accordingly, rafts are defined by the physical properties of the lipid bilayer and function by selective partitioning of membrane lipids and proteins into membrane domains of specific phase behaviour and lipid packing. Here, I will discuss the interplay of these independent principles of protein scaffolds and raft lipid microdomains leading to the generation of biologically functional membrane domains. PMID:12803918

  18. Influenza M2 Transmembrane Domain Senses Membrane Heterogeneity and Enhances Membrane Curvature.

    PubMed

    Ho, Chian Sing; Khadka, Nawal K; She, Fengyu; Cai, Jianfeng; Pan, Jianjun

    2016-07-01

    Targeting host cell membranes by M2 of influenza A virus is important for virus invasion and replication. We study the transmembrane domain of M2 (M2TM) interacting with mica-supported planar bilayers and free-standing giant unilamellar vesicles (GUVs). Using solution atomic force microscopy (AFM), we show that the size of M2TM oligomers is dependent on lipid composition. The addition of M2TM to lipid bilayers containing liquid-ordered (Lo) and liquid-disordered (Ld) phases reveals that M2TM preferentially partitions into the Ld phase; phase-dependent partitioning results in a larger rigidity of the Ld phase. We next use fluorescence microscopy to study the effects of M2TM on phase-coexisting GUVs. In particular, M2TM is found to increase GUVs' miscibility transition temperature Tmix. The augmented thermodynamic stability can be accounted for by considering an enhanced energy barrier of lipid mixing between coexisting phases. Our GUV study also shows that M2TM can elicit an array of vesicle shapes mimicking virus budding. M2TM enhanced membrane curvature is consistent with our AFM data, which show altered membrane rigidity and consequently line tension at domain edges. Together, our results highlight that in addition to conducting protons, M2TM can actively regulate membrane heterogeneity and augment membrane curvature. PMID:27285399

  19. Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

    PubMed

    Schapire, Arnaldo L; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A

    2008-12-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329

  20. High Efficiency Solar Integrated Roof Membrane Product

    SciTech Connect

    Partyka, Eric; Shenoy, Anil

    2013-05-15

    This project was designed to address the Solar Energy Technology Program objective, to develop new methods to integrate photovoltaic (PV) cells or modules within a building-integrated photovoltaic (BIPV) application that will result in lower installed cost as well as higher efficiencies of the encapsulated/embedded PV module. The technology assessment and development focused on the evaluation and identification of manufacturing technologies and equipment capable of producing such low-cost, high-efficiency, flexible BIPV solar cells on single-ply roofing membranes.

  1. Assessing equine sperm-membrane integrity.

    PubMed

    Lagares, M A; Petzoldt, R; Sieme, H; Klug, E

    2000-05-01

    The swelling of cells in a hypo-osmotic medium has been described as an important criterion for assessing the functional integrity of the sperm plasma membrane. The resistance of equine spermatozoa to osmolarity changes was studied by extending 98 semen samples collected from nine stallions in media at five osmolarities (300, 200, 150, 100, and 50 mOsmol l(-1)). The response of the cells was measured by the spermatocrit technique and eosin staining. Spermatocrit determines the increase on spermatozoal volume under hypo-osmotic conditions, a sign of functional integrity of sperm plasma membrane, whereas the eosin staining evaluates the viability of spermatozoa. A significant positive correlation (P<0.01) was observed between spermatocrit values and percentage of eosin-unstained cells. Spermatocrit measurements and eosin staining proved to be useful methods to evaluate the integrity of sperm plasma membrane under hypo-osmotic conditions and could be used as an additional criterion to predict semen preservation ability. PMID:10863971

  2. Solution NMR Structure of Membrane-Integral Diacylglycerol Kinase

    PubMed Central

    Van Horn, Wade D.; Kim, Hak-Jun; Ellis, Charles D.; Hadziselimovic, Arina; Sulistijo, Endah S.; Karra, Murthy D.; Tian, Changlin; Sönnichsen, Frank D.; Sanders, Charles R.

    2009-01-01

    Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer using solution NMR. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK’s three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK’s lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis. PMID:19556511

  3. Pinkbar is an epithelial-specific BAR domain protein that generates planar membrane structures

    SciTech Connect

    Pykäläinen, Anette; Boczkowska, Malgorzata; Zhao, Hongxia; Saarikangas, Juha; Rebowski, Grzegorz; Jansen, Maurice; Hakanen, Janne; Koskela, Essi V.; Peränen, Johan; Vihinen, Helena; Jokitalo, Eija; Salminen, Marjo; Ikonen, Elina; Dominguez, Roberto; Lappalainen, Pekka

    2013-05-29

    Bin/amphipysin/Rvs (BAR)-domain proteins sculpt cellular membranes and have key roles in processes such as endocytosis, cell motility and morphogenesis. BAR domains are divided into three subfamilies: BAR- and F-BAR-domain proteins generate positive membrane curvature and stabilize cellular invaginations, whereas I-BAR-domain proteins induce negative curvature and stabilize protrusions. We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane tubulation but promotes the formation of planar membrane sheets. Structural and mutagenesis analyses reveal that the BAR domain of Pinkbar has a relatively flat lipid-binding interface and that it assembles into sheet-like oligomers in crystals and in solution, which may explain its unique membrane-deforming activity.

  4. Membrane Binding and Self-Association of the Epsin N-Terminal Homology Domain

    PubMed Central

    Lai, Chun-Liang; Jao, Christine C.; Lyman, Edward; Gallop, Jennifer L.; Peter, Brian J.; McMahon, Harvey T.; Langen, Ralf; Voth, Gregory A.

    2012-01-01

    Epsin possesses a conserved epsin N-terminal homology (ENTH) domain that acts as a phosphatidylinositol 4,5-bisphosphate‐lipid‐targeting and membrane‐curvature‐generating element. Upon binding phosphatidylinositol 4,5‐bisphosphate, the N-terminal helix (H0) of the ENTH domain becomes structured and aids in the aggregation of ENTH domains, which results in extensive membrane remodeling. In this article, atomistic and coarse-grained (CG) molecular dynamics (MD) simulations are used to investigate the structure and the stability of ENTH domain aggregates on lipid bilayers. EPR experiments are also reported for systems composed of different ENTH-bound membrane morphologies, including membrane vesicles as well as preformed membrane tubules. The EPR data are used to help develop a molecular model of ENTH domain aggregates on preformed lipid tubules that are then studied by CG MD simulation. The combined computational and experimental approach suggests that ENTH domains exist predominantly as monomers on vesiculated structures, while ENTH domains self-associate into dimeric structures and even higher‐order oligomers on the membrane tubes. The results emphasize that the arrangement of ENTH domain aggregates depends strongly on whether the local membrane curvature is isotropic or anisotropic. The molecular mechanism of ENTH‐domain-induced membrane vesiculation and tubulation and the implications of the epsin's role in clathrin-mediated endocytosis resulting from the interplay between ENTH domain membrane binding and ENTH domain self-association are also discussed. PMID:22922484

  5. Thermoelectric Integrated Membrane Evaporation Subsystem operational improvements

    NASA Technical Reports Server (NTRS)

    Dehner, G. F.; Winkler, H. E.; Reysa, R. P.

    1984-01-01

    A three-man preprototype Thermoelectric Integrated Membrane Evaporation Subsystem (TIMES) has been developed to provide high quality water recovery from waste fluids on extended duration space flights. In the most recent effort, a number of improvements have been made to simplify subsystem operation and increase performance. These modifications include changes to the hollow fiber membrane evaporator, the condensing section of the thermoelectric heat pump, and the electronic controller logic and display. This paper describes the results of the test program that was conducted to evaluate the implemented improvements. In addition, an advanced design concept is discussed that will provide lower electrical power consumption, greater water production capacity, lower weight, and a smaller package than the present subsystem configuration.

  6. Thermoelectric integrated membrane evaporation water recovery technology

    NASA Technical Reports Server (NTRS)

    Roebelen, G. J., Jr.; Winkler, H. E.; Dehner, G. F.

    1982-01-01

    The recently developed Thermoelectric Integrated Membrane Evaporation Subsystem (TIMES) offers a highly competitive approach to water recovery from waste fluids for future on-orbit stations such as the Space Operations Center. Low power, compactness and gravity insensitive operation are featured in this vacuum distillation subsystem that combines a hollow fiber membrane evaporator with a thermoelectric heat pump. The hollow fiber elements provide positive liquid/gas phase control with no moving parts other than pumps and an accumulator, thus solving problems inherent in other reclamation subsystem designs. In an extensive test program, over 850 hours of operation were accumulated during which time high quality product water was recovered from both urine and wash water at an average steady state production rate of 2.2 pounds per hour.

  7. Physiochemical Properties of Aluminum Adjuvants Elicit Differing Reorganization of Phospholipid Domains in Model Membranes.

    PubMed

    Antúnez, Lorena R; Livingston, Andrea; Berkland, Cory; Dhar, Prajnaparamita

    2016-05-01

    Most vaccines contain aluminum adjuvants; however, their exact mechanism of action remains unclear. A novel mechanism by Shi and colleagues proposes aluminum adjuvants may enhance immune activation by binding and reorganizing lipids that are key components of lipid rafts. To better understand the specificity of interaction between aluminum adjuvants and the cell membrane lipids, we present a biophysical study of lipid domain clustering in simple model phospholipid monolayers containing dipalmitoyl-phosphatidylcholine (DPPC) and dioleoyl-phosphatidylcholine (DOPC) exposed to two aluminum adjuvants, Alhydrogel and Adju-Phos. Surface pressure measurements and fluorescence microscopy images verified aluminum adjuvant-induced increase in lipid domain size, even in the key lipid raft components. Additionally, adjuvant induced lipid clustering differed based on the physicochemical properties of the adjuvants. Alhydrogel appeared to reduce monolayer compressibility and insert into the monolayer, while Adju-Phos induced more significant changes in domain size, without compromising the integrity of the monolayer. The Alhydrogel and Adju-Phos-mediated reorganization of phospholipid domains reported here supports the new mechanistic paradigm proposed by Shi and co-workers, and further suggests that lipid clustering is induced even in simple phospholipid membranes. The results present the basis for future exploration into lipid-mediated mechanisms of action for adjuvants. PMID:26998680

  8. The N-Terminal Domain of Bcl-xL Reversibly Binds Membranes in a pH-Dependent Manner†

    PubMed Central

    Thuduppathy, Guruvasuthevan R.; Terrones, Oihana; Craig, Jeffrey W.; Basañez, Gorka; Hill, R. Blake

    2006-01-01

    Bcl-xL regulates apoptosis by maintaining the integrity of the mitochondrial outer membrane by adopting both soluble and membrane-associated forms. The membrane-associated conformation does not require a conserved, C-terminal transmembrane domain and appears to be inserted into the bilayer of synthetic membranes as assessed by membrane permeabilization and critical surface pressure measurements. Membrane association is reversible and is regulated by the cooperative binding of approximately two protons to the protein. Two acidic residues, Glu153 and Asp156, that lie in a conserved hairpin of Bcl-xLΔTM appear to be important in this process on the basis of a 16% increase in the level of membrane association of the double mutant E153Q/D156N. Contrary to that for the wild type, membrane permeabilization for the mutant is not correlated with membrane association. Monolayer surface pressure measurements suggest that this effect is primarily due to less membrane penetration. These results suggest that E153 and D156 are important for the Bcl-xLΔTM conformational change and that membrane binding can be distinct from membrane permeabilization. Taken together, these studies support a model in which Bcl-xL activity is controlled by reversible insertion of its N-terminal domain into the mitochondrial outer membrane. Future studies with Bcl-xL mutants such as E153Q/D156N should allow determination of the relative contributions of membrane binding, insertion, and permeabilization to the regulation of apoptosis. PMID:17128992

  9. Membrane-Tethered Intracellular Domain of Amphiregulin Promotes Keratinocyte Proliferation

    PubMed Central

    Stoll, Stefan W.; Stuart, Philip E.; Lambert, Sylviane; Gandarillas, Alberto; Rittié, Laure; Johnston, Andrew; Elder, James T.

    2016-01-01

    The EGF receptor (EGFR) and its ligands are essential regulators of epithelial biology, which are often amplified in cancer cells. We have previously shown that shRNA-mediated silencing of one of these ligands, amphiregulin (AREG), results in keratinocyte growth arrest that cannot be rescued by soluble extracellular EGFR ligands. To further explore the functional importance of specific AREG domains, we stably transduced keratinocytes expressing tetracycline-inducible AREG-targeted shRNA with lentiviruses expressing silencing-proof, membrane-tethered AREG cytoplasmic and extracellular domains (AREG-CTD and AREG-ECD), as well as full-length AREG precursor (proAREG). Here we show that growth arrest of AREG-silenced keratinocytes occurs in G2/M and is significantly restored by proAREG and AREG-CTD, but not by AREG-ECD. Moreover, the AREG-CTD was sufficient to normalize cell cycle distribution profiles and expression of mitosis-related genes. Our findings uncover an important role of the AREG-CTD in regulating cell division, which may be relevant to tumor resistance to EGFR-directed therapies. PMID:26802239

  10. Localization of membrane-type 1 matrix metalloproteinase in caveolae membrane domains.

    PubMed Central

    Annabi, B; Lachambre, M; Bousquet-Gagnon, N; Pagé, M; Gingras, D; Béliveau, R

    2001-01-01

    Membrane-type 1 matrix metalloproteinase (MT1-MMP) is a membrane-associated MMP that has been recently reported to have a central role in tumour cell invasion. Here we report that both the native and overexpressed recombinant forms of MT1-MMP are highly enriched in low-density Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin 1. Moreover, the MT1-MMP-dependent activation of proMMP-2 induced by concanavalin A and cytochalasin D was correlated with the processing of MT1-MMP to its proteolytically inactive 43 kDa fragment in U-87 glioblastoma and HT-1080 fibrosarcoma tumour cell lines; this processing was also preferentially observed within the caveolar fraction. Interestingly, whereas the expression of caveolin 1 had no effect on the MT1-MMP-dependent activation of proMMP-2, its co-expression with MT1-MMP antagonized the MT1-MMP-increased migratory potential of COS-7 cells. Taken together, our results provide evidence that MT1-MMP is preferentially compartmentalized and proteolytically processed in caveolae of cancer cells. The inhibition of MT1-MMP-dependent cell migration by caveolin 1 also suggests that the localization of MT1-MMP to caveolin-enriched domains might have an important function in the control of its enzymic activity. PMID:11171051

  11. Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment.

    PubMed

    Flores-Canales, Jose C; Vargas-Uribe, Mauricio; Ladokhin, Alexey S; Kurnikova, Maria

    2015-06-01

    Diphtheria toxin translocation (T) domain inserts in lipid bilayers upon acidification of the environment. Computational and experimental studies have suggested that low pH triggers a conformational change of the T-domain in solution preceding membrane binding. The refolded membrane-competent state was modeled to be compact and mostly retain globular structure. In the present work, we investigate how this refolded state interacts with membrane interfaces in the early steps of T-domain's membrane association. Coarse-grained molecular dynamics simulations suggest two distinct membrane-bound conformations of the T-domain in the presence of bilayers composed of a mixture of zwitteronic and anionic phospholipids (POPC:POPG with a 1:3 molar ratio). Both membrane-bound conformations show a common near parallel orientation of hydrophobic helices TH8-TH9 relative to the membrane plane. The most frequently observed membrane-bound conformation is stabilized by electrostatic interactions between the N-terminal segment of the protein and the membrane interface. The second membrane-bound conformation is stabilized by hydrophobic interactions between protein residues and lipid acyl chains, which facilitate deeper protein insertion in the membrane interface. A theoretical estimate of a free energy of binding of a membrane-competent T-domain to the membrane is provided. PMID:25650178

  12. Interaction of the Intermembrane Space Domain of Tim23 Protein with Mitochondrial Membranes*

    PubMed Central

    Bajaj, Rakhi; Munari, Francesca; Becker, Stefan; Zweckstetter, Markus

    2014-01-01

    Tim23 mediates protein translocation into mitochondria. Although inserted into the inner membrane, the dynamic association of its intermembrane space (IMS) domain with the outer membrane promotes protein import. However, little is known about the molecular basis of this interaction. Here, we demonstrate that the IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N terminus. The structure of membrane-bound Tim23IMS is highly dynamic, allowing recognition of both the incoming presequence and other translocase components at the translocation contact. Cardiolipin enhances Tim23 membrane attachment, suggesting that cardiolipin can influence preprotein import. PMID:25349212

  13. Quality control of integral membrane proteins by assembly-dependent membrane integration.

    PubMed

    Feige, Matthias J; Hendershot, Linda M

    2013-08-01

    Cell-surface multiprotein complexes are synthesized in the endoplasmic reticulum (ER), where they undergo cotranslational membrane integration and assembly. The quality control mechanisms that oversee these processes remain poorly understood. We show that less hydrophobic transmembrane (TM) regions derived from several single-pass TM proteins can enter the ER lumen completely. Once mislocalized, they are recognized by the Hsp70 chaperone BiP. In a detailed analysis for one of these proteins, the αβT cell receptor (αβTCR), we show that unassembled ER-lumenal subunits are rapidly degraded, whereas specific subunit interactions en route to the native receptor promote membrane integration of the less hydrophobic TM segments, thereby stabilizing the protein. For the TCR α chain, both complete ER import and subunit assembly depend on the same pivotal residue in its TM region. Thus, membrane integration linked to protein assembly allows cellular quality control of membrane proteins and connects the lumenal ER chaperone machinery to membrane protein biogenesis. PMID:23932713

  14. Evidence that Membrane Insertion of the Cytosolic Domain of Bcl-xL is Governed by an Electrostatic Mechanism

    PubMed Central

    Thuduppathy, Guruvasuthevan R.; Craig, Jeffrey W.; Schon, Victoria Kholodenko Arne; Hill, R. Blake

    2006-01-01

    Signals from different cellular networks are integrated at the mitochondria in the regulation of apoptosis. This integration is controlled by the Bcl-2 proteins, many of which change localization fromthe cytosol to the mitochondrial outer membrane in this regulation. For Bcl-xL, this change in localization reflects the ability to undergo a conformational change from a solution to integral membrane conformation. To characterize this conformational change, structural and thermodynamic measurements were performed in the absence and presence of lipid vesicles with Bcl-xL. A pH-dependent model is proposed for the solution to membrane conformational change that consists of three stable conformations: a solution conformation, a conformation similar to the solution conformation but anchored to the membrane by its C-terminal transmembrane domain, and a membrane conformation that is fully associated with the membrane. This model predicts that the solution to membrane conformational change is independent of the C-terminal trans-membrane domain, which is experimentally demonstrated. The conformational change is associated with changes in secondary and, especially, tertiary structure of the protein, as measured by far and near-UV circular dichroism spectroscopy, respectively. Membrane insertion was distinguished from peripheral association with the membrane by quenching of intrinsic tryptophan fluorescence by acrylamide and brominated lipids. For the cytosolic domain, the free energy of insertion ( ΔGox) into lipid vesicles was determined to be −6.5 k cal mol−1 at pH4.9 by vesicle binding experiments. To test whether electrostatic interactions were significant to this process, the salt dependence of this conformational change was measured and analyzed in terms of Gouy–Chapman theory to estimate an electrostatic contribution of ΔGoel ~−2.5 kcal mol−1 and a non-electrostatic contribution of ΔGonel ~−4.0 kcal mol−1 to the free energy of insertion, ΔGox. Calcium

  15. Crystallization and preliminary X-ray analysis of the periplasmic domain of FliP, an integral membrane component of the bacterial flagellar type III protein-export apparatus

    PubMed Central

    Fukumura, Takuma; Furukawa, Yukio; Kawaguchi, Tatsuya; Saijo-Hamano, Yumiko; Namba, Keiichi; Imada, Katsumi; Minamino, Tohru

    2014-01-01

    The bacterial flagellar proteins are transported via a specific export apparatus to the distal end of the growing structure for their self-assembly. FliP is an essential membrane component of the export apparatus. FliP has an N-terminal signal peptide and is predicted to have four transmembrane (TM) helices and a periplasmic domain (FliPP) between TM-2 and TM-3. In this study, FliPP from Thermotoga maritima (TmFliPP) and its selenomethionine derivative (SeMet-TmFliPP) were purified and crystallized. TmFliPP formed a homotetramer in solution. Crystals of TmFliPP and SeMet-TmFliPP were obtained by the hanging-drop vapour-diffusion technique with 2-methyl-2,4-pentanediol as a precipitant. These two crystals grew in the hexagonal space group P6222 or P6422, with unit-cell parameters a = b = 114.9, c = 193.8 Å. X-ray diffraction data were collected from crystals of TmFliPP and SeMet-TmFliPP to 2.4 and 2.8 Å resolution, respectively. PMID:25195894

  16. On the First Eigenvalues of Free Vibrating Membranes in Conformal Regular Domains

    NASA Astrophysics Data System (ADS)

    Gol'dshtein, V.; Ukhlov, A.

    2016-08-01

    In 1961 G. Polya published a paper about the eigenvalues of vibrating membranes. The "free vibrating membrane" corresponds to the Neumann-Laplace operator in bounded plane domains. In this paper we obtain estimates for the first non-trivial eigenvalue of this operator in a large class of domains that we call conformal regular domains. This class includes convex domains, John domains etc. On the basis of our estimates we conjecture that the eigenvalues of the Neumann- Laplace operator depend on the hyperbolic metrics of plane domains. We propose a new method for the estimates which is based on weighted Poincaré-Sobolev inequalities, obtained by the authors recently.

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

    PubMed

    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. PMID:26884618

  18. Determining the Topology of Integral Membrane Peptides Using EPR Spectroscopy

    PubMed Central

    Inbaraj, Johnson J.; Cardon, Thomas B.; Laryukhin, Mikhail; Grosser, Stuart M.

    2008-01-01

    This paper reports on the development of a new structural biology technique for determining the membrane topology of an integral membrane protein inserted into magnetically aligned phospholipid bilayers (bicelles) using EPR spectroscopy. The nitroxide spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) was attached to the pore-lining transmembrane domain (M2δ) of the nicotinic acetylcholine receptor (AChR) and incorporated into a bicelle. The corresponding EPR spectra revealed hyperfine splittings that were highly dependent on the macroscopic orientation of the bicelles with respect to the static magnetic field. The helical tilt of the peptide can be easily calculated using the hyperfine splittings gleaned from the orientational dependent EPR spectra. A helical tilt of 14° was calculated for the M2δ peptide with respect to the bilayer normal of the membrane, which agrees well with previous 15N solid-state NMR studies. The helical tilt of the peptide was verified by simulating the corresponding EPR spectra using the standardized MOMD approach. This new method is advantageous because: (1) bicelle samples are easy to prepare, (2) the helical tilt can be directly calculated from the orientational-dependent hyperfine splitting in the EPR spectra, and (3) EPR spectroscopy is approximately 1000 fold more sensitive than 15N solid-state NMR spectroscopy; thus, the helical tilt of an integral membrane peptide can be determined with only 100 μg of peptide. The helical tilt can be determined more accurately by placing TOAC spin labels at several positions with this technique. PMID:16848493

  19. Treatment of domain integrals in boundary element methods

    SciTech Connect

    Nintcheu Fata, Sylvain

    2012-01-01

    A systematic and rigorous technique to calculate domain integrals without a volume-fitted mesh has been developed and validated in the context of a boundary element approximation. In the proposed approach, a domain integral involving a continuous or weakly-singular integrand is first converted into a surface integral by means of straight-path integrals that intersect the underlying domain. Then, the resulting surface integral is carried out either via analytic integration over boundary elements or by use of standard quadrature rules. This domain-to-boundary integral transformation is derived from an extension of the fundamental theorem of calculus to higher dimension, and the divergence theorem. In establishing the method, it is shown that the higher-dimensional version of the first fundamental theorem of calculus corresponds to the well-known Poincare lemma. The proposed technique can be employed to evaluate integrals defined over simply- or multiply-connected domains with Lipschitz boundaries which are embedded in an Euclidean space of arbitrary but finite dimension. Combined with the singular treatment of surface integrals that is widely available in the literature, this approach can also be utilized to effectively deal with boundary-value problems involving non-homogeneous source terms by way of a collocation or a Galerkin boundary integral equation method using only the prescribed surface discretization. Sample problems associated with the three-dimensional Poisson equation and featuring the Newton potential are successfully solved by a constant element collocation method to validate this study.

  20. Enrichment of Integral Membrane Proteins for Proteomic Analysis Using Liquid Chromatography-Tandem Mass Spectrometry

    SciTech Connect

    Blonder, Josip; Goshe, Michael B.; Moore, Ronald J.; Pasa-Tolic, Liljiana; Masselon, Christophe D.; Lipton, Mary S.; Smith, Richard D.

    2002-04-01

    Currently, most proteomic studies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents, chaotropes, or reducing reagents that often interfere with electrospray ionization (ESI). To increase the identification of integral membrane proteins by LC-ESI-MS/MS, a sample preparation method combining carbonate extraction and surfactant-free organics solvent-assisted solubilization and proteolysis was developed and used to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic based on their positive grand average of hydropathicity values that covers 15% of the theoretical hydrophobic proteome. Using the PSORT algorithm, 268 identified proteins were recognized as integral membrane proteins covering 21% and 43% of the predicted integral cytoplasmic and outer membrane proteins, respectively. Of the integral cytoplasmic membrane proteins containing four or more predicted transmembrane domains (TMDs), 65% were identified by detecting at least one peptide spanning a TMD using LC-MS/MS. The extensive identification of highly hydrophobic proteins containing multiple TMDs confirms the efficacy of the described sample preparation protocol to isolate and solubilize integral membrane proteins and validates the method for large-scale analysis of bacterial membrane subproteomes using LC-ESI-MS/MS.

  1. Localization of lysobisphosphatidic acid-rich membrane domains in late endosomes.

    PubMed

    Kobayashi, T; Startchev, K; Whitney, A J; Gruenber, J

    2001-03-01

    Late endosomes accumulate internal membranes within the lumen of the organelle. These internal membranes are enriched in the late endosome specific phospholipid, lysobisphosphatidic acid (LBPA). The organization of LBPA-rich membrane domains is not well characterized. Using an LBPA-specific monoclonal antibody (6C4), we show that these membrane domains are not accessible from the cytoplasm. Using fluorescence correlation spectroscopy, we also show that 6C4 only binds sonicated, but not intact, late endosomes, presumably reflecting the release of internal membranes upon endosome rupture. PMID:11347897

  2. Regulation of membrane-shape transitions induced by I-BAR domains.

    PubMed

    Chen, Zhiming; Shi, Zheng; Baumgart, Tobias

    2015-07-21

    I-BAR proteins are well-known actin-cytoskeleton adaptors and have been observed to be involved in the formation of plasma membrane protrusions (filopodia). I-BAR proteins contain an all-helical, crescent-shaped IRSp53-MIM domain (IMD) dimer that is believed to be able to couple with a membrane shape. This coupling could involve the sensing and even the generation of negative plasma membrane curvature. Indeed, the in vitro studies have shown that IMDs can induce inward tubulation of liposomes. While N-BAR domains, which generate positive membrane curvature, have received a considerable amount of attention from both theory and experiments, the mechanisms of curvature coupling through IMDs are comparatively less studied and understood. Here we used a membrane-shape stability assay developed recently in our lab to quantitatively characterize IMD-induced membrane-shape transitions. We determined a membrane-shape stability diagram for IMDs that reveals how membrane tension and protein density can comodulate the generation of IMD-induced membrane protrusions. From comparison to analytical theory, we determine three key parameters that characterize the curvature coupling of IMD. We find that the curvature generation capacity of IMDs is significantly stronger compared to that of endophilin, an N-BAR protein known to be involved in plasma membrane shape transitions. Contrary to N-BAR domains, where amphipathic helix insertion is known to promote its membrane curvature generation, for IMDs we find that amphipathic helices inhibit membrane shape transitions, consistent with the inverse curvature that IMDs generate. Importantly, in both of these types of BAR domains, electrostatic interactions affect membrane-binding capacity, but do not appear to affect the curvature generation capacity of the protein. These two types of BAR domain proteins show qualitatively similar membrane shape stability diagrams, suggesting an underlying ubiquitous mechanism by which peripheral proteins

  3. Integrating the Affective Domain into the Instructional Design Process.

    ERIC Educational Resources Information Center

    Main, Robert G.

    This study begins with a definition of the affective domain and its importance to learning, outlining its impact both in achieving affective behaviors and in facilitating cognitive and psychomotor objectives. The study then develops a model of instructional design that incorporates the affective domain as an integral component. The model combines…

  4. An Integrated Model of Information Literacy, Based upon Domain Learning

    ERIC Educational Resources Information Center

    Thompson, Gary B.; Lathey, Johnathan W.

    2013-01-01

    Introduction. Grounded in Alexander's model of domain learning, this study presents an integrated micro-model of information literacy. It is predicated upon the central importance of domain learning for the development of the requisite research skills by students. Method. The authors reviewed previous models of information literacy and…

  5. Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

    PubMed Central

    Monk, Brian C.; Tomasiak, Thomas M.; Keniya, Mikhail V.; Huschmann, Franziska U.; Tyndall, Joel D. A.; O’Connell, Joseph D.; Cannon, Richard D.; McDonald, Jeffrey G.; Rodriguez, Andrew; Finer-Moore, Janet S.; Stroud, Robert M.

    2014-01-01

    Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance. PMID:24613931

  6. Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer.

    PubMed

    Monk, Brian C; Tomasiak, Thomas M; Keniya, Mikhail V; Huschmann, Franziska U; Tyndall, Joel D A; O'Connell, Joseph D; Cannon, Richard D; McDonald, Jeffrey G; Rodriguez, Andrew; Finer-Moore, Janet S; Stroud, Robert M

    2014-03-11

    Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance. PMID:24613931

  7. ABCA1, ABCG1, and ABCG4 are distributed to distinct membrane meso-domains and disturb detergent-resistant domains on the plasma membrane.

    PubMed

    Sano, Osamu; Ito, Shiho; Kato, Reiko; Shimizu, Yuji; Kobayashi, Aya; Kimura, Yasuhisa; Kioka, Noriyuki; Hanada, Kentaro; Ueda, Kazumitsu; Matsuo, Michinori

    2014-01-01

    ATP-binding cassette A1 (ABCA1), ABCG1, and ABCG4 are lipid transporters that mediate the efflux of cholesterol from cells. To analyze the characteristics of these lipid transporters, we examined and compared their distributions and lipid efflux activity on the plasma membrane. The efflux of cholesterol mediated by ABCA1 and ABCG1, but not ABCG4, was affected by a reduction of cellular sphingomyelin levels. Detergent solubility and gradient density ultracentrifugation assays indicated that ABCA1, ABCG1, and ABCG4 were distributed to domains that were solubilized by Triton X-100 and Brij 96, resistant to Triton X-100 and Brij 96, and solubilized by Triton X-100 but resistant to Brij 96, respectively. Furthermore, ABCG1, but not ABCG4, was colocalized with flotillin-1 on the plasma membrane. The amounts of cholesterol extracted by methyl-β-cyclodextrin were increased by ABCA1, ABCG1, or ABCG4, suggesting that cholesterol in non-raft domains was increased. Furthermore, ABCG1 and ABCG4 disturbed the localization of caveolin-1 to the detergent-resistant domains and the binding of cholera toxin subunit B to the plasma membrane. These results suggest that ABCA1, ABCG1, and ABCG4 are localized to distinct membrane meso-domains and disturb the meso-domain structures by reorganizing lipids on the plasma membrane; collectively, these observations may explain the different substrate profiles and lipid efflux roles of these transporters. PMID:25302608

  8. Nonbilayer lipids affect peripheral and integral membrane proteins via changes in the lateral pressure profile.

    PubMed

    van den Brink-van der Laan, Els; Killian, J Antoinette; de Kruijff, Ben

    2004-11-01

    Nonbilayer lipids can be defined as cone-shaped lipids with a preference for nonbilayer structures with a negative curvature, such as the hexagonal phase. All membranes contain these lipids in large amounts. Yet, the lipids in biological membranes are organized in a bilayer. This leads to the question: what is the physiological role of nonbilayer lipids? Different models are discussed in this review, with a focus on the lateral pressure profile within the membrane. Based on this lateral pressure model, predictions can be made for the effect of nonbilayer lipids on peripheral and integral membrane proteins. Recent data on the catalytic domain of Leader Peptidase and the potassium channel KcsA are discussed in relation to these predictions and in relation to the different models on the function of nonbilayer lipids. The data suggest a general mechanism for the interaction between nonbilayer lipids and membrane proteins via the membrane lateral pressure. PMID:15519321

  9. Membrane Fission Is Promoted by Insertion of Amphipathic Helices and Is Restricted by Crescent BAR Domains

    PubMed Central

    Boucrot, Emmanuel; Pick, Adi; Çamdere, Gamze; Liska, Nicole; Evergren, Emma; McMahon, Harvey T.; Kozlov, Michael M.

    2012-01-01

    Summary Shallow hydrophobic insertions and crescent-shaped BAR scaffolds promote membrane curvature. Here, we investigate membrane fission by shallow hydrophobic insertions quantitatively and mechanistically. We provide evidence that membrane insertion of the ENTH domain of epsin leads to liposome vesiculation, and that epsin is required for clathrin-coated vesicle budding in cells. We also show that BAR-domain scaffolds from endophilin, amphiphysin, GRAF, and β2-centaurin limit membrane fission driven by hydrophobic insertions. A quantitative assay for vesiculation reveals an antagonistic relationship between amphipathic helices and scaffolds of N-BAR domains in fission. The extent of vesiculation by these proteins and vesicle size depend on the number and length of amphipathic helices per BAR domain, in accord with theoretical considerations. This fission mechanism gives a new framework for understanding membrane scission in the absence of mechanoenzymes such as dynamin and suggests how Arf and Sar proteins work in vesicle scission. PMID:22464325

  10. Simultaneous evaluation of plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential in bovine spermatozoa by flow cytometry.

    PubMed

    Kanno, Chihiro; Kang, Sung-Sik; Kitade, Yasuyuki; Yanagawa, Yojiro; Takahashi, Yoshiyuki; Nagano, Masashi

    2016-08-01

    The present study aimed to develop an objective evaluation procedure to estimate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bull spermatozoa simultaneously by flow cytometry. Firstly, we used frozen-thawed semen mixed with 0, 25, 50, 75 or 100% dead spermatozoa. Semen was stained using three staining solutions: SYBR-14, propidium iodide (PI), and phycoerythrin-conjugated peanut agglutinin (PE-PNA), for the evaluation of plasma membrane integrity and acrosomal integrity. Then, characteristics evaluated by flow cytometry and by fluorescence microscopy were compared. Characteristics of spermatozoa (viability and acrosomal integrity) evaluated by flow cytometry and by fluorescence microscopy were found to be similar. Secondly, we attempted to evaluate the plasma membrane integrity, acrosomal integrity, and also mitochondrial membrane potential of spermatozoa by flow cytometry using conventional staining with three dyes (SYBR-14, PI, and PE-PNA) combined with MitoTracker Deep Red (MTDR) staining (quadruple staining). The spermatozoon characteristics evaluated by flow cytometry using quadruple staining were then compared with those of staining using SYBR-14, PI, and PE-PNA and staining using SYBR-14 and MTDR. There were no significant differences in all characteristics (viability, acrosomal integrity, and mitochondrial membrane potential) evaluated by quadruple staining and the other procedures. In conclusion, quadruple staining using SYBR-14, PI, PE-PNA, and MTDR for flow cytometry can be used to evaluate the plasma membrane integrity, acrosomal integrity, and mitochondrial membrane potential of bovine spermatozoa simultaneously. PMID:26369275

  11. Membrane Binding and Modulation of the PDZ Domain of PICK1

    PubMed Central

    Erlendsson, Simon; Madsen, Kenneth Lindegaard

    2015-01-01

    Scaffolding proteins serve to assemble protein complexes in dynamic processes by means of specific protein-protein and protein-lipid binding domains. Many of these domains bind either proteins or lipids exclusively; however, it has become increasingly evident that certain domains are capable of binding both. Especially, many PDZ domains, which are highly abundant protein-protein binding domains, bind lipids and membranes. Here we provide an overview of recent large-scale studies trying to generalize and rationalize the binding patterns as well as specificity of PDZ domains towards membrane lipids. Moreover, we review how these PDZ-membrane interactions are regulated in the case of the synaptic scaffolding protein PICK1 and how this might affect cellular localization and function. PMID:26501328

  12. HIV gp41-Mediated Membrane Fusion Occurs at Edges of Cholesterol-Rich Lipid Domains

    PubMed Central

    Yang, Sung-Tae; Kiessling, Volker; Simmons, James A.; White, Judith M.; Tamm, Lukas K.

    2015-01-01

    Lipid rafts in plasma membranes have emerged as possible platforms for entry of HIV and other viruses into cells. However, how lipid phase heterogeneity contributes to viral entry is little known due to the fine-grained and still poorly understood complexity of biological membranes. We used model systems mimicking HIV envelopes and T-cell membranes and showed that raft-like (Lo phase) lipid domains are necessary and sufficient for efficient membrane targeting and fusion. Interestingly, membrane binding and fusion was low in homogeneous Ld and Lo phase membranes, indicating that lipid phase heterogeneity is essential. The HIV fusion peptide preferentially targeted to Lo/Ld boundary regions and promoted full fusion at the interface between ordered and disordered lipids. Ld phase vesicles proceeded only to hemifusion. Thus, we propose that the edges, but not the areas of raft-like ordered lipid domains are vital for HIV entry and membrane fusion. PMID:25915200

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

    PubMed Central

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

    2006-01-01

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

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

    PubMed

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

    2006-03-01

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

  15. ELASTIC MEMBRANE DEFORMATIONS GOVERN INTERLEAFLET COUPLING OF LIPID-ORDERED DOMAINS

    PubMed Central

    Galimzyanov, Timur R.; Molotkovsky, Rodion J.; Bozdaganyan, Marine E.; Cohen, Fredric S.; Pohl, Peter; Akimov, Sergey A.

    2016-01-01

    The mechanism responsible for domain registration in two membrane leaflets has thus far remained enigmatic. Using continuum elasticity theory, we show that minimum line tension is achieved along the rim between thicker (ordered) and thinner (disordered) domains by shifting the rims in opposing leaflets by a few nanometers relative to each other. Increasing surface tension yields an increase in line tension, resulting in larger domains. Because domain registration is driven by lipid deformation energy, it does not require special lipid components nor interactions at the membrane midplane. PMID:26340212

  16. Elastic Membrane Deformations Govern Interleaflet Coupling of Lipid-Ordered Domains.

    PubMed

    Galimzyanov, Timur R; Molotkovsky, Rodion J; Bozdaganyan, Marine E; Cohen, Fredric S; Pohl, Peter; Akimov, Sergey A

    2015-08-21

    The mechanism responsible for domain registration in two membrane leaflets has thus far remained enigmatic. Using continuum elasticity theory, we show that minimum line tension is achieved along the rim between thicker (ordered) and thinner (disordered) domains by shifting the rims in opposing leaflets by a few nanometers relative to each other. Increasing surface tension yields an increase in line tension, resulting in larger domains. Because domain registration is driven by lipid deformation energy, it does not require special lipid components or interactions at the membrane midplane. PMID:26340212

  17. Integrated system for extraction, purification, and digestion of membrane proteins.

    PubMed

    Liu, Yiying; Yan, Guoquan; Gao, Mingxia; Deng, Chunhui; Zhang, Xiangmin

    2016-05-01

    An integrated system was developed for directly processing living cells into peptides of membrane proteins. Living cells were directly injected into the system and cracked in a capillary column by ultrasonic treatment. Owing to hydrophilicity for broken pieces of the cell membrane, the obtained membranes were retained in a well-designed bi-filter. While cytoplasm proteins were eluted from the bi-filter, the membranes were dissolved and protein released by flushing 4 % SDS buffer through the bi-filter. The membrane proteins were subsequently transferred into a micro-reactor and covalently bound in the reactor for purification and digestion. As the system greatly simplified the whole pretreatment processes and minimized both sample loss and contamination, it could be used to analyze the membrane proteome samples of thousand-cell-scales with acceptable reliability and stability. We totally identified 1348 proteins from 5000 HepG2 cells, 615 of which were annotated as membrane proteins. In contrast, with conventional method, only 233 membrane proteins were identified. It is adequately demonstrated that the integrated system shows promising practicability for the membrane proteome analysis of small amount of cells. Graphical Abstract The legend of online abstract figure is (a) schematic illustration of membrane proteins extraction, purification and digestion from living cells; (b) diagrammatic sketch of the automatic integrated membrane proteome analysis system. PMID:26922343

  18. Closed membrane shapes with attached BAR domains subject to external force of actin filaments.

    PubMed

    Mesarec, Luka; Góźdź, Wojciech; Iglič, Veronika Kralj; Kralj, Samo; Iglič, Aleš

    2016-05-01

    Membrane deformations induced by attached BAR superfamily domains could trigger or facilitate the growth of plasma membrane protrusions. The BAR domain family consists of BAR, F-BAR and I-BAR domains, each enforcing a different local curvature when attached to the membrane surface. Our theoretical study mainly focuses on the role of I-BAR in the membrane tubular deformations generated or stabilised by actin filaments. The influence of the area density of membrane attached BAR domains and their intrinsic curvature on the closed membrane shapes (vesicles) was investigated numerically. We derived an analytical approximative expression for the critical relative area density of BARs at which the membrane tubular protrusions on vesicles are most prominent. We have shown that the BARs with a higher intrinsic curvature induce thinner and longer cylindrical protrusions. The average orientation of the membrane attached BARs is altered when the vesicle shape is subjected to external force of growing actin rod-like structure inside a vesicle. The average orientation angle of membrane attached BARs may indicate whether the actin filaments are just stabilising the protrusion or generating it by stretching the vesicle. PMID:26854580

  19. Membrane Anchoring of Aminoacyl-tRNA Synthetases by Convergent Acquisition of a Novel Protein Domain*

    PubMed Central

    Olmedo-Verd, Elvira; Santamaría-Gómez, Javier; Ochoa de Alda, Jesús A. G.; Ribas de Pouplana, Lluis; Luque, Ignacio

    2011-01-01

    Four distinct aminoacyl-tRNA synthetases (aaRSs) found in some cyanobacterial species contain a novel protein domain that bears two putative transmembrane helices. This CAAD domain is present in glutamyl-, isoleucyl-, leucyl-, and valyl-tRNA synthetases, the latter of which has probably recruited the domain more than once during evolution. Deleting the CAAD domain from the valyl-tRNA synthetase of Anabaena sp. PCC 7120 did not significantly modify the catalytic properties of this enzyme, suggesting that it does not participate in its canonical tRNA-charging function. Multiple lines of evidence suggest that the function of the CAAD domain is structural, mediating the membrane anchorage of the enzyme, although membrane localization of aaRSs has not previously been described in any living organism. Synthetases containing the CAAD domain were localized in the intracytoplasmic thylakoid membranes of cyanobacteria and were largely absent from the plasma membrane. The CAAD domain was necessary and apparently sufficient for protein targeting to membranes. Moreover, localization of aaRSs in thylakoids was important under nitrogen limiting conditions. In Anabaena, a multicellular filamentous cyanobacterium often used as a model for prokaryotic cell differentiation, valyl-tRNA synthetase underwent subcellular relocation at the cell poles during heterocyst differentiation, a process also dependent on the CAAD domain. PMID:21965654

  20. Approximation method to compute domain related integrals in structural studies

    NASA Astrophysics Data System (ADS)

    Oanta, E.; Panait, C.; Raicu, A.; Barhalescu, M.; Axinte, T.

    2015-11-01

    Various engineering calculi use integral calculus in theoretical models, i.e. analytical and numerical models. For usual problems, integrals have mathematical exact solutions. If the domain of integration is complicated, there may be used several methods to calculate the integral. The first idea is to divide the domain in smaller sub-domains for which there are direct calculus relations, i.e. in strength of materials the bending moment may be computed in some discrete points using the graphical integration of the shear force diagram, which usually has a simple shape. Another example is in mathematics, where the surface of a subgraph may be approximated by a set of rectangles or trapezoids used to calculate the definite integral. The goal of the work is to introduce our studies about the calculus of the integrals in the transverse section domains, computer aided solutions and a generalizing method. The aim of our research is to create general computer based methods to execute the calculi in structural studies. Thus, we define a Boolean algebra which operates with ‘simple’ shape domains. This algebraic standpoint uses addition and subtraction, conditioned by the sign of every ‘simple’ shape (-1 for the shapes to be subtracted). By ‘simple’ shape or ‘basic’ shape we define either shapes for which there are direct calculus relations, or domains for which their frontiers are approximated by known functions and the according calculus is carried out using an algorithm. The ‘basic’ shapes are linked to the calculus of the most significant stresses in the section, refined aspect which needs special attention. Starting from this idea, in the libraries of ‘basic’ shapes, there were included rectangles, ellipses and domains whose frontiers are approximated by spline functions. The domain triangularization methods suggested that another ‘basic’ shape to be considered is the triangle. The subsequent phase was to deduce the exact relations for the

  1. Valorization of artichoke wastewaters by integrated membrane process.

    PubMed

    Conidi, C; Cassano, A; Garcia-Castello, E

    2014-01-01

    In this work an integrated membrane system was developed on laboratory scale to fractionate artichoke wastewaters. In particular, a preliminary ultrafiltration (UF) step, based on the use of hollow fibre membranes, was investigated to remove suspended solids from an artichoke extract. The clarified solution was then submitted to a nanofiltration (NF) step. Two different 2.5 × 21 in. spiral-wound membranes (Desal DL and NP030) with different properties were investigated. Both membranes showed a high rejection towards the phenolic compounds analysed (chlorogenic acid, cynarin and apigenin-7-O-glucoside) and, consequently, towards the total antioxidant activity (TAA). On the other hand, the Desal DL membrane was characterized by a high rejection towards sugar compounds (glucose, fructose and sucrose) (100%) when compared with the NP030 membrane (4.02%). The performance of selected membranes in terms of permeate flux, fouling index and water permeability recovery was also evaluated. On the base of experimental results, an integrated membrane process for the fractionation of artichoke wastewaters was proposed. This conceptual process design permitted to obtain different valuable products: a retentate fraction (from the NP030 membrane) enriched in phenolic compounds suitable for nutraceutical, cosmeceutical or food application; a retentate fraction (from the Desal DL membrane), enriched in sugar compounds, of interest for food applications; a clear permeate (from the Desal DL membrane) which can be reused as process water or for membrane cleaning. PMID:24125635

  2. Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol

    DOE PAGESBeta

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-04-22

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize themore » cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.« less

  3. Sphingolipid domains in the plasma membranes of fibroblasts are not enriched with cholesterol

    SciTech Connect

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-04-22

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. As a result, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton.

  4. Sphingolipid Domains in the Plasma Membranes of Fibroblasts Are Not Enriched with Cholesterol*

    PubMed Central

    Frisz, Jessica F.; Klitzing, Haley A.; Lou, Kaiyan; Hutcheon, Ian D.; Weber, Peter K.; Zimmerberg, Joshua; Kraft, Mary L.

    2013-01-01

    The plasma membranes of mammalian cells are widely expected to contain domains that are enriched with cholesterol and sphingolipids. In this work, we have used high-resolution secondary ion mass spectrometry to directly map the distributions of isotope-labeled cholesterol and sphingolipids in the plasma membranes of intact fibroblast cells. Although acute cholesterol depletion reduced sphingolipid domain abundance, cholesterol was evenly distributed throughout the plasma membrane and was not enriched within the sphingolipid domains. Thus, we rule out favorable cholesterol-sphingolipid interactions as dictating plasma membrane organization in fibroblast cells. Because the sphingolipid domains are disrupted by drugs that depolymerize the cells actin cytoskeleton, cholesterol must instead affect the sphingolipid organization via an indirect mechanism that involves the cytoskeleton. PMID:23609440

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

    PubMed Central

    Owen, Dylan M.; Gaus, Katharina

    2013-01-01

    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. PMID:24376453

  6. Membrane activity of the phospholipase C-δ1 pleckstrin homology (PH) domain

    PubMed Central

    2005-01-01

    PH-PLCδ1 [the PH domain (pleckstrin homology domain) of PLCδ1 (phospholipase C-δ1)] is among the best-characterized phosphoinositide-binding domains. PH-PLCδ1 binds with high specificity to the headgroup of PtdIns(4,5)P2, but little is known about its interfacial properties. In the present study, we show that PH-PLCδ1 is also membrane-active and can insert significantly into PtdIns(4,5)P2-containing monolayers at physiological (bilayer-equivalent) surface pressures. However, this membrane activity appears to involve interactions distinct from those that target PH-PLCδ1 to the PtdIns(4,5)P2 headgroup. Whereas the majority of PtdIns(4,5)P2-bound PH-PLCδ1 can be displaced by adding excess of soluble headgroup [Ins(1,4,5)P3], membrane activity of PH-PLCδ1 cannot. PH-PLCδ1 differs from other phosphoinositide-binding domains in that its membrane insertion does not require that the phosphoinositide-binding site be occupied. Significant monolayer insertion remains when the phosphoinositide-binding site is mutated, and PH-PLCδ1 can insert into monolayers that contain no PtdIns(4,5)P2 at all. Our results suggest a model in which reversible membrane binding of PH-PLCδ1, mediated by PtdIns(4,5)P2 or other acidic phospholipids, occurs without membrane insertion. Accumulation of the PH domain at the membrane surface enhances the efficiency of insertion, but does not significantly affect its extent, whereas the presence of phosphatidylethanolamine and cholesterol in the lipid mixture promotes the extent of insertion. This is the first report of membrane activity in an isolated PH domain and has implications for understanding the membrane targeting by this common type of domain. PMID:15755258

  7. Investigating the role of viral integral membrane proteins in promoting the assembly of nepovirus and comovirus replication factories

    PubMed Central

    Sanfaçon, Hélène

    2013-01-01

    Formation of plant virus membrane-associated replication factories requires the association of viral replication proteins and viral RNA with intracellular membranes, the recruitment of host factors and the modification of membranes to form novel structures that house the replication complex. Many viruses encode integral membrane proteins that act as anchors for the replication complex. These hydrophobic proteins contain transmembrane domains and/or amphipathic helices that associate with the membrane and modify its structure. The comovirus Co-Pro and NTP-binding (NTB, putative helicase) proteins and the cognate nepovirus X2 and NTB proteins are among the best characterized plant virus integral membrane replication proteins and are functionally related to the picornavirus 2B, 2C, and 3A membrane proteins. The identification of membrane association domains and analysis of the membrane topology of these proteins is discussed. The evidence suggesting that these proteins have the ability to induce membrane proliferation, alter the structure and integrity of intracellular membranes, and modulate the induction of symptoms in infected plants is also reviewed. Finally, areas of research that need further investigation are highlighted. PMID:23439982

  8. Phase Separation on Bicontinuous Cubic Membranes: Symmetry Breaking, Reentrant, and Domain Faceting

    NASA Astrophysics Data System (ADS)

    Paillusson, Fabien; Pennington, Matthew R.; Kusumaatmaja, Halim

    2016-07-01

    We study the phase separation of binary lipid mixtures that form bicontinuous cubic phases. The competition between the nonuniform Gaussian membrane curvature and line tension leads to a very rich phase diagram, where we observe symmetry breaking of the membrane morphologies and reentrant phenomena due to the formation of bridges between segregated domains. Upon increasing the line tension contribution, we also find faceting of lipid domains that we explain using a simple argument based on the symmetry of the underlying surface and topology.

  9. Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability[W

    PubMed Central

    Schapire, Arnaldo L.; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A.

    2008-01-01

    Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca2+-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca2+-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness. PMID:19088329

  10. Study of Raft Domains in Model Membrane of DPPC/PE/Cholesterol

    NASA Astrophysics Data System (ADS)

    Lor, Chai; Hirst, Linda

    2010-10-01

    Raft domains in bilayer membrane are thought to play an important role in many cell functions such as cell signaling or trans-membrane protein activation. Here we use a model membrane consisting of DPPC/PE/cholesterol to examine the structure of membrane rafts and phase interactions. In particular we are interested in lipids containing the highly polyunsaturated fatty acid DHA. We use both atomic force microscopy (AFM) and fluorescence microscopy to obtain information on the structural properties of raft regions and track cholesterol. As expected, we find phase separation of raft regions between saturated and unsaturated lipids. Moreover, we find that the roughness of the domains change with varying cholesterol concentration possibly due to overpacking. This model study provides further understanding of the role of cholesterol in bilayer membrane leading towards a better knowledge of cell membranes.

  11. Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

    PubMed Central

    Takahashi, Satoe

    2007-01-01

    The Rho-type GTPase Cdc42 is a central regulator of eukaryotic cell polarity and signal transduction. In budding yeast, Cdc42 regulates polarity and mitogen-activated protein (MAP) kinase signaling in part through the PAK-family kinase Ste20. Activation of Ste20 requires a Cdc42/Rac interactive binding (CRIB) domain, which mediates its recruitment to membrane-associated Cdc42. Here, we identify a separate domain in Ste20 that interacts directly with membrane phospholipids and is critical for its function. This short region, termed the basic-rich (BR) domain, can target green fluorescent protein to the plasma membrane in vivo and binds PIP2-containing liposomes in vitro. Mutation of basic or hydrophobic residues in the BR domain abolishes polarized localization of Ste20 and its function in both MAP kinase–dependent and independent pathways. Thus, Cdc42 binding is required but is insufficient; instead, direct membrane binding by Ste20 is also required. Nevertheless, phospholipid specificity is not essential in vivo, because the BR domain can be replaced with several heterologous lipid-binding domains of varying lipid preferences. We also identify functionally important BR domains in two other yeast Cdc42 effectors, Gic1 and Gic2, suggesting that cooperation between protein–protein and protein–membrane interactions is a prevalent mechanism during Cdc42-regulated signaling and perhaps for other dynamic localization events at the cell cortex. PMID:17914055

  12. Fragmentation of Integral Membrane Proteins in the Gas Phase

    PubMed Central

    2015-01-01

    Integral membrane proteins (IMPs) are of great biophysical and clinical interest because of the key role they play in many cellular processes. Here, a comprehensive top down study of 152 IMPs and 277 soluble proteins from human H1299 cells including 11 087 fragments obtained from collisionally activated dissociation (CAD), 6452 from higher-energy collisional dissociation (HCD), and 2981 from electron transfer dissociation (ETD) shows their great utility and complementarity for the identification and characterization of IMPs. A central finding is that ETD is ∼2-fold more likely to cleave in soluble regions than threshold fragmentation methods, whereas the reverse is observed in transmembrane domains with an observed ∼4-fold bias toward CAD and HCD. The location of charges just prior to dissociation is consistent with this directed fragmentation: protons remain localized on basic residues during ETD but easily mobilize along the backbone during collisional activation. The fragmentation driven by these protons, which is most often observed in transmembrane domains, both is of higher yield and occurs over a greater number of backbone cleavage sites. Further, while threshold dissociation events in transmembrane domains are on average 10.1 (CAD) and 9.2 (HCD) residues distant from the nearest charge site (R, K, H, N-terminus), fragmentation is strongly influenced by the N- or C-terminal position relative to that site: the ratio of observed b- to y-fragments is ∼1:3 if the cleavage occurs >7 residues N-terminal and ∼3:1 if it occurs >7 residues C-terminal to the nearest basic site. Threshold dissociation products driven by a mobilized proton appear to be strongly dependent on not only relative position of a charge site but also N- or C-terminal directionality of proton movement. PMID:24689519

  13. BamA POTRA Domain Interacts with a Native Lipid Membrane Surface.

    PubMed

    Fleming, Patrick J; Patel, Dhilon S; Wu, Emilia L; Qi, Yifei; Yeom, Min Sun; Sousa, Marcelo Carlos; Fleming, Karen G; Im, Wonpil

    2016-06-21

    The outer membrane of Gram-negative bacteria is an asymmetric membrane with lipopolysaccharides on the external leaflet and phospholipids on the periplasmic leaflet. This outer membrane contains mainly β-barrel transmembrane proteins and lipidated periplasmic proteins (lipoproteins). The multisubunit protein β-barrel assembly machine (BAM) catalyzes the insertion and folding of the β-barrel proteins into this membrane. In Escherichia coli, the BAM complex consists of five subunits, a core transmembrane β-barrel with a long periplasmic domain (BamA) and four lipoproteins (BamB/C/D/E). The BamA periplasmic domain is composed of five globular subdomains in tandem called POTRA motifs that are key to BAM complex formation and interaction with the substrate β-barrel proteins. The BAM complex is believed to undergo conformational cycling while facilitating insertion of client proteins into the outer membrane. Reports describing variable conformations and dynamics of the periplasmic POTRA domain have been published. Therefore, elucidation of the conformational dynamics of the POTRA domain in full-length BamA is important to understand the function of this molecular complex. Using molecular dynamics simulations, we present evidence that the conformational flexibility of the POTRA domain is modulated by binding to the periplasmic surface of a native lipid membrane. Furthermore, membrane binding of the POTRA domain is compatible with both BamB and BamD binding, suggesting that conformational selection of different POTRA domain conformations may be involved in the mechanism of BAM-facilitated insertion of outer membrane β-barrel proteins. PMID:27332128

  14. A nodal domain theorem for integrable billiards in two dimensions

    SciTech Connect

    Samajdar, Rhine; Jain, Sudhir R.

    2014-12-15

    Eigenfunctions of integrable planar billiards are studied — in particular, the number of nodal domains, ν of the eigenfunctions with Dirichlet boundary conditions are considered. The billiards for which the time-independent Schrödinger equation (Helmholtz equation) is separable admit trivial expressions for the number of domains. Here, we discover that for all separable and non-separable integrable billiards, ν satisfies certain difference equations. This has been possible because the eigenfunctions can be classified in families labelled by the same value of mmodkn, given a particular k, for a set of quantum numbers, m,n. Further, we observe that the patterns in a family are similar and the algebraic representation of the geometrical nodal patterns is found. Instances of this representation are explained in detail to understand the beauty of the patterns. This paper therefore presents a mathematical connection between integrable systems and difference equations. - Highlights: • We find that the number of nodal domains of eigenfunctions of integrable, planar billiards satisfy a class of difference equations. • The eigenfunctions labelled by quantum numbers (m,n) can be classified in terms of mmodkn. • A theorem is presented, realising algebraic representations of geometrical patterns exhibited by the domains. • This work presents a connection between integrable systems and difference equations.

  15. Affinity Labeling of Highly Hydrophobic Integral Membrane Proteins for Proteome-Wide Analysis

    SciTech Connect

    Goshe, Michael B.; Blonder, Josip; Smith, Richard D.

    2003-03-01

    The ability to identify and quantify integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and derivatize these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified of which 40 were integral membrane proteins containing from 1 to 9 mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g. ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g. pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.

  16. Raft-Like Membrane Domains in Pathogenic Microorganisms

    PubMed Central

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

    2016-01-01

    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 packed in a liquid-ordered state, commonly known as lipid rafts, are believed to exist. While less studied in bacterial cells, reports on the presence of sterol or protein-mediated microdomains in bacterial cell membranes are also appearing with increasing frequency. Recent efforts have been focused on addressing the biophysical and biochemical properties of lipid rafts. However, most studies have been focused on synthetic membranes, mammalian cells, and/or model, non-pathogenic microorganisms. Much less 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 the developing field of microdomains in pathogenic bacteria. The current literature on the structure and function and of microdomains is reviewed and the potential role of microdomains in growth, pathogenesis, and drug resistance of pathogens are discussed. Better insight into the structure and function of membrane microdomains in pathogenic microorganisms might lead to a better understanding of the process of pathogenesis and development of raft-mediated approaches for new methods of therapy. PMID:26015285

  17. Supramolecular organization of the human N-BAR domain in shaping the sarcolemma membrane.

    PubMed

    Daum, Bertram; Auerswald, Andrea; Gruber, Tobias; Hause, Gerd; Balbach, Jochen; Kühlbrandt, Werner; Meister, Annette

    2016-06-01

    The 30kDa N-BAR domain of the human Bin1 protein is essential for the generation of skeletal muscle T-tubules. By electron cryo-microscopy and electron cryo-tomography with a direct electron detector, we found that Bin1-N-BAR domains assemble into scaffolds of low long-range order that form flexible membrane tubules. The diameter of the tubules closely matches the curved shape of the N-BAR domain, which depends on the composition of the target membrane. These insights are fundamental to our understanding of T-tubule formation and function in human skeletal muscle. PMID:27016283

  18. Virus removal and integrity in aged RO membranes.

    PubMed

    Pype, Marie-Laure; Donose, Bogdan C; Martí, Llucia; Patureau, Dominique; Wery, Nathalie; Gernjak, Wolfgang

    2016-03-01

    Membrane ageing reduces the quality of the filtered water. Therefore, in order to warrant public health, monitoring membrane performances are of utmost importance. Reverse osmosis (RO) membranes are generally used to remove viruses and salt. However, there is no detailed study demonstrating the impact of aged membrane on the rejection of viruses and of membrane integrity indicators. In this paper, the impact of hypochlorite induced RO ageing on the rejection of a virus surrogate (MS2 phage) and four membrane integrity indicators (salt, dissolved organic matter, rhodamine WT and sulphate) was evaluated. Hypochlorite exposure was either active (under filtration) or passive (soaking), and the changes of the membrane surface chemistry were characterised using several autopsy techniques. Under this accelerated ageing condition, the introduction of chlorine in the membrane chemistry and the breakage of amide bonds caused an increase of the water permeability and a decrease of the virus surrogate's and indicators' rejection. Ageing resulted in a more negatively charged membrane and also in a higher hydrophobicity, which lead to the adsorption of MS2 phage. Despite severe physical membrane damage leading to a reduction of salt rejection to 1.2 log (94%), the minimum rejection of MS2 phage stayed on or above 4 log. PMID:26724450

  19. Yeast cell wall integrity sensors form specific plasma membrane microdomains important for signalling.

    PubMed

    Kock, Christian; Arlt, Henning; Ungermann, Christian; Heinisch, Jürgen J

    2016-09-01

    The cell wall integrity (CWI) pathway of the yeast Saccharomyces cerevisiae relies on the detection of cell surface stress by five sensors (Wsc1, Wsc2, Wsc3, Mid2, Mtl1). Each sensor contains a single transmembrane domain and a highly mannosylated extracellular region, and probably detects mechanical stress in the cell wall or the plasma membrane. We here studied the distribution of the five sensors at the cell surface by using fluorescently tagged variants in conjunction with marker proteins for established membrane compartments. We find that each of the sensors occupies a specific microdomain at the plasma membrane. The novel punctate 'membrane compartment occupied by Wsc1' (MCW) shows moderate overlap with other Wsc-type sensors, but not with those of the Mid-type sensors or other established plasma membrane domains. We further observed that sensor density and formation of the MCW compartment depends on the cysteine-rich head group near the N-terminus of Wsc1. Yet, signalling capacity depends more on the sensor density in the plasma membrane than on clustering within its microcompartment. We propose that the MCW microcompartment provides a quality control mechanism for retaining functional sensors at the plasma membrane to prevent them from endocytosis. PMID:27337501

  20. Integral estimates for differentiable functions on irregular domains

    SciTech Connect

    Besov, Oleg V

    2011-02-11

    Integral representations for functions and their partial derivatives in terms of some fixed system of partial derivatives are constructed on irregular domains in a Euclidean space. Embedding theorems for Sobolev-type spaces into a Lebesgue space are established and the norms of the derivatives are estimated. Bibliography: 17 titles.

  1. Quantifying the lateral lipid domain properties in erythrocyte ghost membranes using EPR-spectra decomposition.

    PubMed

    Arsov, Zoran; Schara, Milan; Strancar, Janez

    2002-07-01

    Using EPR spectroscopy a typical lateral domain structure was detected in the membranes of spin-labeled bovine erythrocyte ghosts. The spectral parameters were determined by decomposing the EPR spectrum into three spectral components and tuned by a hybrid-evolutionary-optimization method. In our experiments the lateral domain structure and its properties were influenced by the variation in the temperature and by the addition of n-butanol. The specific responses of the particular domain types were detected. For the most-ordered domain type a break was seen in the temperature dependence of its order parameter, while the order parameters of the two less-ordered domain types exhibited a continuous decrease. Below the break-point temperature the alcohol-induced membrane fluidity variation is mainly a consequence of the change in the proportions of the least- and the most-ordered domain type and not the change of the domain-type ordering or dynamics (with n-butanol concentration). On the other hand, the fluidity variation above the break-point temperature arises from both types of changes. Interestingly, the proportion of the domain type that has its order parameter between that of the least- and the most-ordered domain type remains almost constant with concentration as well as with temperature, which implies its stability. Such characterization of the lateral membrane domain structure could be beneficial when considering the lipid-protein interactions, because it can be assumed that the activity of the membrane-bound enzyme depends on the properties of the particular domain type. PMID:12202132

  2. Molecular assemblies and membrane domains in multivesicular endosome dynamics

    SciTech Connect

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

    2009-05-15

    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.

  3. Molecular assemblies and membrane domains in multivesicular endosome dynamics.

    PubMed

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

    2009-05-15

    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. PMID:19133258

  4. Solid-state NMR Study of the YadA Membrane-Anchor Domain in the Bacterial Outer Membrane.

    PubMed

    Shahid, Shakeel A; Nagaraj, Madhu; Chauhan, Nandini; Franks, Trent W; Bardiaux, Benjamin; Habeck, Michael; Orwick-Rydmark, Marcella; Linke, Dirk; van Rossum, Barth-J

    2015-10-19

    MAS-NMR was used to study the structure and dynamics at ambient temperatures of the membrane-anchor domain of YadA (YadA-M) in a pellet of the outer membrane of E. coli in which it was expressed. YadA is an adhesin from the pathogen Yersinia enterocolitica that is involved in interactions with the host cell, and it is a model protein for studying the autotransport process. Existing assignments were sucessfully transferred to a large part of the YadA-M protein in the E. coli lipid environment by using (13) C-(13) C DARR and PDSD spectra at different mixing times. The chemical shifts in most regions of YadA-M are unchanged relative to those in microcrystalline YadA-M preparations from which a structure has previously been solved, including the ASSA region that is proposed to be involved in transition-state hairpin formation for transport of the soluble domain. Comparisons of the dynamics between the microcrystalline and membrane-embedded samples indicate greater flexibility of the ASSA region in the outer-membrane preparation at physiological temperatures. This study will pave the way towards MAS-NMR structure determination of membrane proteins, and a better understanding of functionally important dynamic residues in native membrane environments. PMID:26332158

  5. delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains.

    PubMed

    Bourova, Lenka; Kostrnova, Alexandra; Hejnova, Lucie; Moravcova, Zuzana; Moon, Hyo-Eun; Novotny, Jiri; Milligan, Graeme; Svoboda, Petr

    2003-04-01

    Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor

  6. Slow Relaxation of Shape and Orientational Texture in Membrane Gel Domains.

    PubMed

    Jeppesen, Jonas Camillus; Solovyeva, Vita; Brewer, Jonathan R; Johannes, Ludger; Hansen, Per Lyngs; Simonsen, Adam Cohen

    2015-11-24

    Gel domains in lipid bilayers are structurally more complex than fluid domains. Growth dynamics can lead to noncircular domains with a heterogeneous orientational texture. Most model membrane studies involving gel domain morphology and lateral organization assume the domains to be static. Here we show that rosette shaped gel domains, with heterogeneous orientational texture and a central topological defect, after early stage growth, undergo slow relaxation. On a time scale of days to weeks domains converge to circular shapes and approach uniform texture. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) enriched gel domains are grown by cooling 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC):DPPC bilayers into the solid-liquid phase coexistence region and are visualized with fluorescence microscopy. The relaxation of individual domains is quantified through image analysis of time-lapse image series. We find a shape relaxation mechanism which is inconsistent with Ostwald ripening and coalescence as observed in membrane systems with coexisting liquid phases. Moreover, domain texture changes in parallel with the changes in domain shape, and selective melting and growth of particular subdomains cause the texture to become more uniform. We propose a relaxation mechanism based on relocation of lipids from high-energy lattice positions, through evaporation-condensation and edge diffusion, to low-energy positions. The relaxation process is modified significantly by binding Shiga toxin, a bacterial toxin from Shigella dysenteriae, to the membrane surface. Binding alters the equilibrium shape of the gel domains from circular to eroded rosettes with disjointed subdomains. This observation may be explained by edge diffusion while evaporation-condensation is restricted, and it provides further support for the proposed overall relaxation mechanism. PMID:26501924

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

    PubMed

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

    2016-04-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  9. Specific effect of polyunsaturated fatty acids on the cholesterol-poor membrane domain in a model membrane.

    PubMed

    Onuki, Yoshinori; Hagiwara, Chihiro; Sugibayashi, Ko; Takayama, Kozo

    2008-08-01

    To understand more fully the effect of polyunsaturated fatty acids (PUFAs) on lipid bilayers, we investigated the effects of treatment with fatty acids on the properties of a model membrane. Three kinds of liposomes comprising dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC), and cholesterol (Ch) were used as the model membrane, and the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and detergent insolubility were determined. Characterization of the liposomes clarified that DPPC, DPPC/Ch, and DPPC/DOPC/Ch existed as solid-ordered phase (L beta), liquid-ordered phase (l o), and a mixture of l o and liquid-disordered phase (L alpha) membranes at room temperature. Treatment with unsaturated fatty acids such as oleic acid (OA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) markedly decreased the fluorescence anisotropy value and detergent insolubility. PUFAs and OA had different effects on the model membranes. In DPPC liposomes, the most prominent change was induced by PUFAs, whereas, in DPPC/Ch and DPPC/DOPC/Ch liposomes, OA had a stronger effect than PUFAs. The effect of PUFAs was strongly affected by the amount of Ch in the membrane, which confirmed a specific effect of PUFAs on the Ch-poor membrane domain. We further explored the effect of fatty acids dispersed in a water-in-oil-in-water multiple emulsion and found that unsaturated fatty acids acted on the membranes even when incorporated in emulsion form. These findings suggest that treatment with PUFAs increases the segregation of ordered and disordered phase domains in membranes. PMID:18670110

  10. Kinetics of Endophilin N-BAR Domain Dimerization and Membrane Interactions*

    PubMed Central

    Capraro, Benjamin R.; Shi, Zheng; Wu, Tingting; Chen, Zhiming; Dunn, Joanna M.; Rhoades, Elizabeth; Baumgart, Tobias

    2013-01-01

    The recruitment to plasma membrane invaginations of the protein endophilin is a temporally regulated step in clathrin-mediated endocytosis. Endophilin is believed to sense or stabilize membrane curvature, which in turn likely depends on the dimeric structure of the protein. The dynamic nature of the membrane association and dimerization of endophilin is thus functionally important and is illuminated herein. Using subunit exchange Förster resonance energy transfer (FRET), we determine dimer dissociation kinetics and find a dimerization equilibrium constant orders of magnitude lower than previously published values. We characterize N-BAR domain membrane association kinetics under conditions where the dimeric species predominates, by stopped flow, observing prominent electrostatic sensitivity of membrane interaction kinetics. Relative to membrane binding, we find that protein monomer/dimer species equilibrate with far slower kinetics. Complementary optical microscopy studies reveal strikingly slow membrane dissociation and an increase of dissociation rate constant for a construct lacking the amphipathic segment helix 0 (H0). We attribute the slow dissociation kinetics to higher-order protein oligomerization on the membrane. We incorporate our findings into a kinetic scheme for endophilin N-BAR membrane binding and find a significant separation of time scales for endophilin membrane binding and subsequent oligomerization. This separation may facilitate the regulation of membrane trafficking phenomena. PMID:23482561

  11. Quantifying Membrane Curvature Generation of Drosophila Amphiphysin N-BAR Domains

    PubMed Central

    Heinrich, Michael C.; Capraro, Benjamin R.; Tian, Aiwei; Isas, Jose M.; Langen, Ralf; Baumgart, Tobias

    2012-01-01

    Biological membrane functions are coupled to membrane curvature, the regulation of which often involves membrane-associated proteins. The membrane-binding N-terminal amphipathic helix-containing BIN/Amphiphysin/Rvs (N-BAR) domain of amphiphysin is implicated in curvature generation and maintenance. Improving the mechanistic understanding of membrane curvature regulation by N-BAR domains requires quantitative experimental characterization. We have measured tube pulling force modulation by the N-BAR domain of Drosophila amphiphysin (DA-N-BAR) bound to tubular membranes pulled from micropipette-aspirated giant vesicles. We observed that fluorescently-labeled DA-N-BAR showed significantly higher protein density on tubules compared to the connected low-curvature vesicle membrane. Furthermore, we found the equilibrium tube pulling force to be systematically dependent on the aqueous solution concentration of DA-N-BAR, thereby providing the first quantitative assessment of spontaneous curvature generation. At sufficiently high protein concentrations, pulled tubes required no external force to maintain mechanical equilibrium, in agreement with the qualitative spontaneous tubulation previously reported for amphiphysin. PMID:23772271

  12. INTRAOPERATIVE SPECTRAL DOMAIN OPTICAL COHERENCE TOMOGRAPHY IMAGING AFTER INTERNAL LIMITING MEMBRANE PEELING IN IDIOPATHIC EPIRETINAL MEMBRANE WITH CONNECTING STRANDS

    PubMed Central

    NAM, DONG HEUN; DESOUZA, PHILIP J.; HAHN, PAUL; TAI, VINCENT; SEVILLA, MONICA B.; TRAN-VIET, DU; CUNEFARE, DAVID; FARSIU, SINA; IZATT, JOSEPH A.; TOTH, CYNTHIA A.

    2015-01-01

    Purpose To report the intraoperative optical coherence tomography findings in idiopathic epiretinal membrane (ERM) with connecting strands and to describe the postoperative outcomes. Methods A retrospective, case series study within a prospective observational intraoperative optical coherence tomography imaging study was performed. Epiretinal membranes with connecting strands were characterized on preoperative spectral domain optical coherence tomography images and assessed against corresponding intraoperative (after internal limiting membrane [ILM] peeling) and postoperative spectral domain optical coherence tomography images. Results Eleven locations of the connecting strands in 7 eyes were studied. The connecting strands had visible connections from the inner retinal surface to the ERM in all locations, and the reflectivity was moderate in 8 locations and high in 3 locations. After ERM and ILM peeling, disconnected strands were identified in all of the intraoperative optical coherence tomography images. The reflectivity of the remaining intraoperative strands was higher than that of the preoperative lesions and appeared as “finger-like” and branching projections. The remaining disconnected lesions were contiguous with the inner retinal layers. Postoperatively, the intraoperative lesions disappeared completely in all locations, and recurrent formation of ERM was not identified in any eyes. Conclusion In ERM eyes with connecting strands, intraoperative spectral domain optical coherence tomography imaging showed moderately to highly reflective sub-ILM finger-like lesions that persist immediately after membrane and ILM peeling. Postoperatively, the hyperreflective lesions disappeared spontaneously without localized nerve fiber layer loss. The sub-ILM connecting strands may represent glial retinal attachments. PMID:25829349

  13. The nanoscale organization of signaling domains at the plasma membrane.

    PubMed

    Griffié, Juliette; Burn, Garth; Owen, Dylan M

    2015-01-01

    In this chapter, we present an overview of the role of the nanoscale organization of signaling domains in regulating key cellular processes. In particular, we illustrate the importance of protein and lipid nanodomains as triggers and mediators of cell signaling. As particular examples, we summarize the state of the art of understanding the role of nanodomains in the mounting of an immune response, cellular adhesion, intercellular communication, and cell proliferation. Thus, this chapter underlines the essential role the nanoscale organization of key signaling proteins and lipid domains. We will also see how nanodomains play an important role in the lifecycle of many pathogens relevant to human disease and therefore illustrate how these structures may become future therapeutic targets. PMID:26015282

  14. Model lipid bilayer with facile diffusion of lipids and integral membrane proteins.

    PubMed

    Wang, Tingting; Ingram, Colin; Weisshaar, James C

    2010-07-01

    A model membrane system is formed by the rupture of giant unilamellar vesicles (GUVs) onto a passivating layer comprising a PEG polymer cushion anchored in a lipid bilayer supported on glass. The novel use of pH-dependent electrostatic interactions between NeutrAvidin in the passivating layer and anionic lipids in the GUV drives vesicle rupture. The resulting "GUV pancakes" are single, planar lipid bilayer patches whose diameters vary from approximately 20 to 50 microm. The pancakes have several potential advantages for the in vitro study of protein-lipid interactions and integral membrane protein function. All components are commercially available. The pancakes resist nonspecific binding of vesicles containing protein. Both lipids and integral membrane proteins exhibit good lateral mobility in the GUV pancakes, as evidenced by single-particle tracking (SPT) of the DiD double-tailed fluorescent probe and of the integral membrane protein syntaxin-1A, labeled with AlexaFluor 633 (AF633-Syx). At least 80% of both probes exhibit free, homogeneous diffusion with a diffusion coefficient of approximately 5.5 microm(2) s(-1), which is more than 10 times faster than diffusion in a GUV pancake supported on bare glass. Atomic force microscopy (AFM) suggests that the polymer cushion has a height of approximately 4 nm. The mobility of a large fraction of the AF633-Syx probe suggests that even integral membrane proteins with large domains on both sides of the lipid bilayer should exhibit free diffusion within a GUV pancake. PMID:20459075

  15. Detergent-free domain isolated from Xenopus egg plasma membrane with properties similar to those of detergent-resistant membranes.

    PubMed

    Luria, Ayala; Vegelyte-Avery, Vaida; Stith, Brad; Tsvetkova, Nelly M; Wolkers, Willem F; Crowe, John H; Tablin, Fern; Nuccitelli, Richard

    2002-11-01

    Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs. PMID:12403620

  16. Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains

    PubMed Central

    Yang, Zhengrong; Wang, Chi; Zhou, Qingxian; An, Jianli; Hildebrandt, Ellen; Aleksandrov, Luba A; Kappes, John C; DeLucas, Lawrence J; Riordan, John R; Urbatsch, Ina L; Hunt, John F; Brouillette, Christie G

    2014-01-01

    Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification. PMID:24652590

  17. Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains.

    PubMed

    Yang, Zhengrong; Wang, Chi; Zhou, Qingxian; An, Jianli; Hildebrandt, Ellen; Aleksandrov, Luba A; Kappes, John C; DeLucas, Lawrence J; Riordan, John R; Urbatsch, Ina L; Hunt, John F; Brouillette, Christie G

    2014-06-01

    Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification. PMID:24652590

  18. Interconnect modeling using integrated time-domain and frequency-domain techniques

    SciTech Connect

    You, Hong; Yeh, Chune-Sin; Gadepally, B.

    1995-12-31

    This paper presents an integrated time- and-frequency-domain technique for characterization and modeling of parasitic effects associated with interconnects. This technique enables direct measurements of critical transient as well as frequency responses of interconnects; accurate and efficient SPICE model extraction for coupled lines; and cross-domain verification of the measured data as well as the extracted models. To illustrate its application this technique is applied to characterize and extract the equivalent circuit model of the I/O bus on a real-world printed circuit board.

  19. Membrane Domains Based on Ankyrin and Spectrin Associated with Cell–Cell Interactions

    PubMed Central

    Bennett, Vann; Healy, Jane

    2009-01-01

    Nodes of Ranvier and axon initial segments of myelinated nerves, sites of cell–cell contact in early embryos and epithelial cells, and neuromuscular junctions of skeletal muscle all perform physiological functions that depend on clustering of functionally related but structurally diverse ion transporters and cell adhesion molecules within microdomains of the plasma membrane. These specialized cell surface domains appeared at different times in metazoan evolution, involve a variety of cell types, and are populated by distinct membrane-spanning proteins. Nevertheless, recent work has shown that these domains all share on their cytoplasmic surfaces a membrane skeleton comprised of members of the ankyrin and spectrin families. This review will summarize basic features of ankyrins and spectrins, and will discuss emerging evidence that these proteins are key players in a conserved mechanism responsible for assembly and maintenance of physiologically important domains on the surfaces of diverse cells. PMID:20457566

  20. Mechanistic similarities in docking of the FYVE and PX domains to phosphatidylinositol 3-phosphate containing membranes

    PubMed Central

    Kutateladze, Tatiana G.

    2007-01-01

    Phosphatidylinositol 3-phosphate [PtdIns(3)P], a phospholipid produced by PI 3-kinases in early endosomes and multivesicular bodies, often serves as a marker of endosomal membranes. PtdIns(3)P recruits and activates effector proteins containing the FYVE or PX domain and therefore regulates a variety of biological processes including endo- and exocytosis, membrane trafficking, protein sorting, signal transduction and cytoskeletal rearrangement. Structures and PtdIns(3)P binding modes of several FYVE and PX domains have recently been characterized, unveiling the molecular basis underlying multiple cellular functions of these proteins. Here, structural and functional aspects and current mechanisms of the multivalent membrane anchoring by the FYVE and PX domains are reviewed and compared. PMID:17707914

  1. Xenon and Other Volatile Anesthetics Change Domain Structure in Model Lipid Raft Membranes

    PubMed Central

    Weinrich, Michael; Worcester, David L.

    2014-01-01

    Inhalation anesthetics have been in clinical use for over 160 years, but the molecular mechanisms of action continue to be investigated. Direct interactions with ion channels received much attention after it was found that anesthetics do not change the structure of homogeneous model membranes. However, it was recently found that halothane, a prototypical anesthetic, changes domain structure of a binary lipid membrane. The noble gas xenon is an excellent anesthetic and provides a pivotal test of the generality of this finding, extended to ternary lipid raft mixtures. We report that xenon and conventional anesthetics change the domain equilibrium in two canonical ternary lipid raft mixtures. These findings demonstrate a membrane-mediated mechanism whereby inhalation anesthetics can affect the lipid environment of trans-membrane proteins. PMID:24299622

  2. Muscle specific kinase: organiser of synaptic membrane domains.

    PubMed

    Ghazanfari, Nazanin; Fernandez, Kristine J; Murata, Yui; Morsch, Marco; Ngo, Shyuan T; Reddel, Stephen W; Noakes, Peter G; Phillips, William D

    2011-03-01

    Muscle Specific Kinase (MuSK) is a transmembrane tyrosine kinase vital for forming and maintaining the mammalian neuromuscular junction (NMJ: the synapse between motor nerve and skeletal muscle). MuSK expression switches on during skeletal muscle differentiation. MuSK then becomes restricted to the postsynaptic membrane of the NMJ, where it functions to cluster acetylcholine receptors (AChRs). The expression, activation and turnover of MuSK are each regulated by signals from the motor nerve terminal. MuSK forms the core of an emerging signalling complex that can be acutely activated by neural agrin (N-agrin), a heparin sulfate proteoglycan secreted from the nerve terminal. MuSK activation initiates complex intracellular signalling events that coordinate the local synthesis and assembly of synaptic proteins. The importance of MuSK as a synapse organiser is highlighted by cases of autoimmune myasthenia gravis in which MuSK autoantibodies can deplete MuSK from the postsynaptic membrane, leading to complete disassembly of the adult NMJ. PMID:20974278

  3. Critical fluctuations in the domain structure of lipid membranes

    NASA Astrophysics Data System (ADS)

    Halstenberg, S.; Schrader, W.; Das, P.; Bhattacharjee, J. K.; Kaatze, U.

    2003-03-01

    Between 100 kHz and 2 GHz ultrasonic attenuation spectra of two aqueous solutions of vesicles from 1,2-dimyristoyl-L-3-phosphatidylcholine have been measured at 13 temperatures around the main phase transition temperature of the membranes. The spectra are analyzed in terms of an asymptotic high frequency background contribution and three relaxation terms. Two of these terms can be represented by a discrete relaxation time, respectively, the other one extends over a significantly broader frequency range than a Debye-type relaxation term. It was found to nicely follow the predictions of the Bhattacharjee-Ferrell model of three-dimensional critical fluctuations. This finding has been additionally verified by measurements of the scaling function and by an analysis of the relaxation rate of order parameter fluctuations following from the fit of the experimental scaling function data to the theoretical form. Theoretical arguments are presented to indicate why the three-dimensional theory applies so well to the quasi-two-dimensional membrane system.

  4. Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes.

    PubMed

    Parasassi, T; Gratton, E; Yu, W M; Wilson, P; Levi, M

    1997-06-01

    Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan. The GP function measures the relative water content of the membrane. In the present study we discuss images obtained with polarized laser excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes. PMID:9168019

  5. Regulation of Rac1 translocation and activation by membrane domains and their boundaries

    PubMed Central

    Moissoglu, Konstadinos; Kiessling, Volker; Wan, Chen; Hoffman, Brenton D.; Norambuena, Andres; Tamm, Lukas K.; Schwartz, Martin Alexander

    2014-01-01

    ABSTRACT The activation of Rac1 and related Rho GTPases involves dissociation from Rho GDP-dissociation inhibitor proteins and translocation to membranes, where they bind effectors. Previous studies have suggested that the binding of Rac1 to membranes requires, and colocalizes with, cholesterol-rich liquid-ordered (lo) membrane domains (lipid rafts). Here, we have developed a fluorescence resonance energy transfer (FRET) assay that robustly detects Rac1 membrane targeting in living cells. Surprisingly, FRET with acceptor constructs that were targeted to either raft or non-raft areas indicated that Rac1 was present in both regions. Functional studies showed that Rac1 localization to non-raft regions decreased GTP loading as a result of inactivation by GTPase-activating proteins. In vitro, Rac1 translocation to supported lipid bilayers also required lo domains, yet Rac1 was concentrated in the liquid-disordered (ld) phase. Single-molecule analysis demonstrated that translocation occurred preferentially at lo–ld boundaries. These results, therefore, suggest that Rac1 translocates to the membrane at domain boundaries, then diffuses into raft and non-raft domains, which controls interactions. These findings resolve discrepancies in our understanding of Rac biology and identify novel mechanisms by which lipid rafts modulate Rho GTPase signaling. PMID:24695858

  6. Phase Separation on Bicontinuous Cubic Membranes: Symmetry Breaking, Reentrant, and Domain Faceting.

    PubMed

    Paillusson, Fabien; Pennington, Matthew R; Kusumaatmaja, Halim

    2016-07-29

    We study the phase separation of binary lipid mixtures that form bicontinuous cubic phases. The competition between the nonuniform Gaussian membrane curvature and line tension leads to a very rich phase diagram, where we observe symmetry breaking of the membrane morphologies and reentrant phenomena due to the formation of bridges between segregated domains. Upon increasing the line tension contribution, we also find faceting of lipid domains that we explain using a simple argument based on the symmetry of the underlying surface and topology. PMID:27517794

  7. LdFlabarin, a New BAR Domain Membrane Protein of Leishmania Flagellum

    PubMed Central

    Thonnus, Magali; Salin, Bénédicte; Boissier, Fanny; Blancard, Corinne; Sauvanet, Cécile; Metzler, Christelle; Espiau, Benoît; Sahin, Annelise; Merlin, Gilles

    2013-01-01

    During the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein. PMID:24086735

  8. Gramicidin Induce Local Non-Uniform Distribution of Lipids in Multi-Component Membrane Domains

    NASA Astrophysics Data System (ADS)

    Mao, Yu; Hussain, Fazle; Huang, Juyang

    2015-03-01

    In lipid membranes, gramicidin form trans-membrane channels that are specific for monovalent cations. We performed Molecular Dynamics simulations of gramicidin in coexisting liquid-ordered (Lo) and liquid disordered (Ld) domains using GROMACS. The lipid compositions of Lo and Ld domains are DOPC/DSPC/Cholesterol = 6.5/52.6/40.9 and 74.4/10.6/15, respectively. In the Ld domain, the membrane thickness matches the hydrophobic length of gramicidin quite well, and water molecules can diffuse through the gramicidin channels. However, in the Lo lipid domain, the bilayer thickness is far greater than the hydrophobic length of gramicidin and majority of gramicidin do not form conducting channel. The simulation result explained our experimental finding that gramicidin partition favorably into the Ld domains. The calculated radial distribution functions of lipids indicate that gramicidin recruit a layer of short DOPC surrounding each protein and keep cholesterol and taller DSPC away from the protein-bilayer interface. Our result indicates that membrane proteins are capable of inducing non-uniform distributions of lipids and creating a local bilayer environment, which favors protein function.

  9. Opening of holes in liposomal membranes is induced by proteins possessing the FERM domain.

    PubMed

    Takeda, Shuichi; Saitoh, Akihiko; Furuta, Mayumi; Satomi, Nao; Ishino, Atsushi; Nishida, Gakushi; Sudo, Hiroaki; Hotani, Hirokazu; Takiguchi, Kingo

    2006-09-22

    The destabilization of vesicles caused by interactions between lipid bilayers and proteins was studied by direct, real-time observation using high-intensity dark-field microscopy. We previously reported that talin, a cytoskeletal submembranous protein, can reversibly open stable large holes in giant liposomes made of neutral and acidic phospholipids. Talin and other proteins belonging to the band 4.1 superfamily have the FERM domain at their N-terminal and interact with lipid membranes via that domain. Here, we observed that band 4.1, ezrin and moesin, members of the band 4.1 superfamily, are also able to open stable holes in liposomes. However, truncation of their C-terminal domains, which can interact with the N-terminal FERM domain, impaired their hole opening activities. Oligomeric states of ezrin affected the capability of the membrane hole formation. Phosphatidylinositol bisphosphate (PIP2), which binds to the FERM domain and disrupts the interaction between the N and C termini of the band 4.1 superfamily, down-regulates their membrane opening activity. These results suggest that the intermolecular interaction plays a key role in the observed membrane hole formation. PMID:16934293

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

    PubMed Central

    Tran, Jason; Magenau, Astrid; Rodriguez, Macarena; Rentero, Carles; Royo, Teresa; Enrich, Carlos; Thomas, Shane R.; Grewal, Thomas; Gaus, Katharina

    2016-01-01

    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. PMID:26977592

  11. Nano-domain formation in charged membranes: Beyond the Debye-Hückel approximation

    NASA Astrophysics Data System (ADS)

    Okamoto, Ryuichi; Shimokawa, Naofumi; Komura, Shigeyuki

    2016-04-01

    We investigate the microphase separation in a membrane composed of charged lipids, by taking into account explicitly the electrostatic potential and the ion densities in the surrounding solvent. While the overall (membrane and solvent) charge neutrality is assumed, the membrane can have a non-zero net charge. The static structure factor in the homogeneous state is analytically obtained without using the Debye-Hückel approximation and is found to have a peak at an intermediate wave number. For a binary membrane composed of anionic and neutral lipids, the characteristic wave number corresponds to a scale from several to tens of nanometers. Our numerical calculation further predicts the existence of nano-domains in charged membranes.

  12. Tracking Cholesterol/Sphingomyelin-Rich Membrane Domains with the Ostreolysin A-mCherry Protein

    PubMed Central

    Skočaj, Matej; Resnik, Nataša; Grundner, Maja; Ota, Katja; Rojko, Nejc; Hodnik, Vesna; Anderluh, Gregor; Sobota, Andrzej; Maček, Peter; Veranič, Peter; Sepčić, Kristina

    2014-01-01

    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. PMID:24664106

  13. Membrane translocation assay based on proteolytic cleavage: Application to diphtheria toxin T domain

    PubMed Central

    Rodnin, Mykola V.; Ladokhin, Alexey S.

    2014-01-01

    The function of diphtheria toxin translocation (T) domain is to transfer the catalytic domain across the endosomal membrane upon acidification. The goal of this study was to develop and apply an in vitro functional assay for T domain activity, suitable for investigation of structure-function relationships of translocation across lipid bilayers of various compositions. Traditionally, T domain activity in vitro is estimated by measuring either conductance in planar lipid bilayers or the release of fluorescent markers from lipid vesicles. While an in vivo cell death assay is the most relevant to physiological function, it cannot be applied to studying the effects of pH or membrane lipid composition on translocation. Here we suggest an assay based on cleavage of the N-terminal part of T domain upon translocation into protease-loaded vesicles. A series of control experiment was used to confirm that cleavage occurs inside the vesicle and not as the result of vesicle disruption. Translocation of the N-terminus of the T domain is shown to require the presence of a critical fraction of anionic lipids, which is consistent with our previous biophysical measurements of insertion. Application of the proposed assay to a series of T domain mutants correlated well with the results of cytotoxicity assay. PMID:25291602

  14. Groundnut Bud Necrosis Virus Encoded NSm Associates with Membranes via Its C-Terminal Domain

    PubMed Central

    Singh, Pratibha; Indi, Shantinath S.; Savithri, Handanahal S.

    2014-01-01

    Groundnut Bud Necrosis Virus (GBNV) is a tripartite ambisense RNA plant virus that belongs to serogroup IV of Tospovirus genus. Non-Structural protein-m (NSm), which functions as movement protein in tospoviruses, is encoded by the M RNA. In this communication, we demonstrate that despite the absence of any putative transmembrane domain, GBNV NSm associates with membranes when expressed in E. coli as well as in N. benthamiana. Incubation of refolded NSm with liposomes ranging in size from 200–250 nm resulted in changes in the secondary and tertiary structure of NSm. A similar behaviour was observed in the presence of anionic and zwitterionic detergents. Furthermore, the morphology of the liposomes was found to be modified in the presence of NSm. Deletion of coiled coil domain resulted in the inability of in planta expressed NSm to interact with membranes. Further, when the C-terminal coiled coil domain alone was expressed, it was found to be associated with membrane. These results demonstrate that NSm associates with membranes via the C-terminal coiled coil domain and such an association may be important for movement of viral RNA from cell to cell. PMID:24919116

  15. Nucleocytoplasmic transport in the midzone membrane domain controls yeast mitotic spindle disassembly

    PubMed Central

    Lucena, Rafael; Dephoure, Noah; Gygi, Steve P.; Kellogg, Douglas R.; Tallada, Victor A.

    2015-01-01

    During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast. PMID:25963819

  16. SMP-domain proteins at membrane contact sites: Structure and function.

    PubMed

    Reinisch, Karin M; De Camilli, Pietro

    2016-08-01

    SMP-domains are found in proteins that localize to membrane contact sites. Elucidation of the properties of these proteins gives clues as to the molecular bases underlying processes that occur at such sites. Described here are recent discoveries concerning the structure, function, and regulation of the Extended-Synaptotagmin proteins and ERMES complex subunits, SMP-domain proteins at endoplasmic reticulum (ER)-plasma membrane and ER-mitochondrial contacts, respectively. They act as tethers contributing to the architecture of these sites and as lipid transporters that convey glycerolipids between apposed membranes. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26686281

  17. Endogenous sphingomyelin segregates into submicrometric domains in the living erythrocyte membrane.

    PubMed

    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

    2014-07-01

    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 (125)I-lysenin* binding to erythrocytes upon SM depletion by SMase. The (125)I-lysenin* binding isotherm indicated saturation at 3.5 × 10(6) molecules/RBC, i.e., ∼3% of SM coverage. Nonsaturating lysenin* concentration also labeled sub-micrometric 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

  18. MALDI Tissue Profiling of Integral Membrane Proteins from Ocular Tissues

    PubMed Central

    Thibault, Danielle B.; Gillam, Christopher J.; Grey, Angus C.; Han, Jun; Schey, Kevin L.

    2008-01-01

    MALDI tissue profiling and imaging have become valuable tools for rapid, direct analysis of tissues to investigate spatial distributions of proteins, potentially leading to an enhanced understanding of the molecular basis of disease. Sample preparation methods developed to date for these techniques produce protein expression profiles from predominantly hydrophilic, soluble proteins. The ability to obtain information about the spatial distribution of integral membrane proteins is critical to more fully understand their role in physiological processes, including transport, adhesion, and signaling. In this communication, a sample preparation method for direct tissue profiling of integral membrane proteins is presented. Spatially resolved profiles for the abundant lens membrane proteins aquaporin 0 (AQP0) and MP20, and the retinal membrane protein opsin, were obtained using this method. MALDI tissue profiling results were validated by analysis of dissected tissue prepared by traditional membrane protein processing methods. Furthermore, direct tissue profiling of lens membrane proteins revealed aged related post-translational modifications, as well as a novel modification that had not been detected using conventional tissue homogenization methods. PMID:18396059

  19. Damping identification in frequency domain using integral method

    NASA Astrophysics Data System (ADS)

    Guo, Zhiwei; Sheng, Meiping; Ma, Jiangang; Zhang, Wulin

    2015-03-01

    A new method for damping identification of linear system in frequency domain is presented, by using frequency response function (FRF) with integral method. The FRF curve is firstly transformed to other type of frequency-related curve by changing the representations of horizontal and vertical axes. For the newly constructed frequency-related curve, integral is conducted and the area forming from the new curve is used to determine the damping. Three different methods based on integral are proposed in this paper, which are called FDI-1, FDI-2 and FDI-3 method, respectively. For a single degree of freedom (Sdof) system, the formulated relation of each method between integrated area and loss factor is derived theoretically. The numeral simulation and experiment results show that, the proposed integral methods have high precision, strong noise resistance and are very stable in repeated measurements. Among the three integral methods, FDI-3 method is the most recommended because of its higher accuracy and simpler algorithm. The new methods are limited to linear system in which modes are well separated, and for closely spaced mode system, mode decomposition process should be conducted firstly.

  20. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane

    PubMed Central

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J.

    2015-01-01

    The organization of proteins and lipids in the plasma membrane has been subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here, we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase, nor result in any enrichment of nanoscopic ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane. PMID:25897971

  1. GPI-anchored proteins do not reside in ordered domains in the live cell plasma membrane.

    PubMed

    Sevcsik, Eva; Brameshuber, Mario; Fölser, Martin; Weghuber, Julian; Honigmann, Alf; Schütz, Gerhard J

    2015-01-01

    The organization of proteins and lipids in the plasma membrane has been the subject of a long-lasting debate. Membrane rafts of higher lipid chain order were proposed to mediate protein interactions, but have thus far not been directly observed. Here we use protein micropatterning combined with single-molecule tracking to put current models to the test: we rearranged lipid-anchored raft proteins (glycosylphosphatidylinositol(GPI)-anchored-mGFP) directly in the live cell plasma membrane and measured the effect on the local membrane environment. Intriguingly, this treatment does neither nucleate the formation of an ordered membrane phase nor result in any enrichment of nanoscopic-ordered domains within the micropatterned regions. In contrast, we find that immobilized mGFP-GPIs behave as inert obstacles to the diffusion of other membrane constituents without influencing their membrane environment over distances beyond their physical size. Our results indicate that phase partitioning is not a fundamental element of protein organization in the plasma membrane. PMID:25897971

  2. Membranes for the Sulfur-Iodine Integrated Laboratory Scale Demonstration

    SciTech Connect

    Frederick F. Stewart

    2007-08-01

    INL has developed polymeric membrane-based chemical separations to enable the thermochemical production of hydrogen. Major activities included studies of sulfuric acid concentration membranes, hydriodic acid concentration membranes, SO2/O2 separation membranes, potential applications of a catalyst reactor system for the decomposition of HI, and evaluation of the chemical separation needs for alternate thermochemical cycles. Membranes for the concentration of sulfuric acid were studied using pervaporation. The goal of this task was to offer the sulfur-iodine (S-I) and the hybrid sulfur (HyS) cycles a method to concentrate the sulfuric acid containing effluent from the decomposer without boiling. In this work, sulfuric acid decomposer effluent needs to be concentrated from ~50 % acid to 80 %. This task continued FY 2006 efforts to characterize water selective membranes for use in sulfuric acid concentration. In FY 2007, experiments were conducted to provide specific information, including transmembrane fluxes, separation factors, and membrane durability, necessary for proper decision making on the potential inclusion of this process into the S-I or HyS Integrated Laboratory Scale demonstration.

  3. Efficient integration of a realistic two-dimensional cardiac tissue model by domain decomposition.

    PubMed

    Quan, W; Evans, S J; Hastings, H M

    1998-03-01

    The size of realistic cardiac tissue models has been limited by their high computational demands. In particular, the Luo-Rudy phase II membrane model, used to simulate a thin sheet of ventricular tissue with arrays of coupled ventricular myocytes, is usually limited to 100 x 100 arrays. We introduce a new numerical method based on domain decomposition and a priority queue integration scheme which reduces the computational cost by a factor of 3-17. In the standard algorithm all the nodes advance with the same time step delta t, whose size is limited by the time scale of activation. However, at any given time, many regions may be inactive and do not require the same small delta t and consequent extensive computations. Hence, adjusting delta t locally is a key factor in improving computational efficiency, since most of the computing time is spent calculating ionic currents. This paper proposes an efficient adaptive numerical scheme for integrating a two-dimensional (2-D) propagation model, by incorporating local adjustments of delta t. In this method, alternating direction Cooley-Dodge and Rush-Larsen methods were used for numerical integration. Between consecutive integrations over the whole domain using an implicit method, the model was spatially decomposed into many subdomains, and delta t adjusted locally. The Euler method was used for numerical integration in the subdomains. Local boundary values were determined from the boundary mesh elements of the neighboring subdomains using linear interpolation. Because delta t was defined locally, a priority queue was used to store and order next update times for each subdomain. The subdomain with the earliest update time was given the highest priority and advanced first. This new method yielded stable solutions with relative errors less than 1% and reduced computation time by a factor of 3-17 and will allow much larger (e.g., 500 x 500) models based on realistic membrane kinetics and realistic dimensions to simulate

  4. Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.

    PubMed

    Mason, R Preston; Jacob, Robert F

    2015-02-01

    Lipid oxidation leads to endothelial dysfunction, inflammation, and foam cell formation during atherogenesis. Glucose also contributes to lipid oxidation and promotes pathologic changes in membrane structural organization, including the development of cholesterol crystalline domains. In this study, we tested the comparative effects of eicosapentaenoic acid (EPA), an omega-3 fatty acid indicated for the treatment of very high triglyceride (TG) levels, and other TG-lowering agents (fenofibrate, niacin, and gemfibrozil) on lipid oxidation in human low-density lipoprotein (LDL) as well as membrane lipid vesicles prepared in the presence of glucose (200 mg/dL). We also examined the antioxidant effects of EPA in combination with atorvastatin o-hydroxy (active) metabolite (ATM). Glucose-induced changes in membrane structural organization were measured using small angle x-ray scattering approaches and correlated with changes in lipid hydroperoxide (LOOH) levels. EPA was found to inhibit LDL oxidation in a dose-dependent manner (1.0-10.0 µM) and was distinguished from the other TG-lowering agents, which had no significant effect as compared to vehicle treatment alone. Similar effects were observed in membrane lipid vesicles exposed to hyperglycemic conditions. The antioxidant activity of EPA, as observed in glucose-treated vesicles, was significantly enhanced in combination with ATM. Glucose treatment produced highly-ordered, membrane-restricted, cholesterol crystalline domains, which correlated with increased LOOH levels. Of the agents tested in this study, only EPA inhibited glucose-induced cholesterol domain formation. These data demonstrate that EPA, at pharmacologic levels, inhibits hyperglycemia-induced changes in membrane lipid structural organization through a potent antioxidant mechanism associated with its distinct, physicochemical interactions with the membrane bilayer. PMID:25449996

  5. Photonic integration in indium-phosphide membranes on silicon (IMOS)

    NASA Astrophysics Data System (ADS)

    van der Tol, Jos; Pello, Josselin; Bhat, Shrivatsa; Jiao, Yuqing; Heiss, Dominik; Roelkens, Gunther; Ambrosius, Huub; Smit, Meint

    2014-03-01

    A new photonic integration technique is presented, based on the use of an indium phosphide membrane on top of a silicon chip. This can provide electronic chips (CMOS) with an added optical layer (IMOS) for resolving the communication bottleneck. A major advantage of InP is the possibility to integrate passive and active components (SOAs, lasers) in a single membrane. In this paper we describe progress achieved in both the passive and active components. For the passive part of the circuit we succeeded to bring the propagation loss of our circuits close to the values obtained with silicon; we achieved propagation loss as low as 3.3 dB/cm through optimization of the lithography and the introduction of C60 (fullerene) in an electro resist. Further we report the smallest polarisation converter reported for membrane waveguides ( <10 μm) with low-loss (< 1 dB from 1520- 1550 nm), > 95% polarisation conversion efficiency over the whole C-band and tolerant fabrication. We also demonstrate an InP-membrane wavelength demultiplexer with a loss of 2.8 dB, a crosstalk level of better than 18 dB and a uniformity over the 8 channels of better than 1.2 dB. For the integration of active components we are testing a twin guide integration scheme. We present our design based on optical and electrical simulations and the fabrication techniques.

  6. AHEAD: Integrated Activities in the High Energy Astrophysics Domain

    NASA Astrophysics Data System (ADS)

    Piro, Luigi; Natalucci, Lorenzo; Ahead Consortium

    2015-09-01

    AHEAD (Integrated Activities in the High Energy Astrophysics Domain) is a forthcoming project approved in the framework of the European Horizon 2020 program (Research Infrastructures for High Energy Astrophysics). The overall objective of AHEAD is to integrate national efforts in high-energy Astrophysics and to promote the domain at the European level, to keep its community at the cutting edge of science and technology and ensure that space observatories for high-energy astrophysics, with particular regard to Athena, are at the state of the art. AHEAD will integrate key research infrastructures for on-ground test and calibration of space-based sensors and electronics and promote their coordinated use. In parallel, the best facilities for data analysis of high-energy astrophysical observatories will be made available to the European community. The technological development will focus on the improvement of selected critical technologies, background modeling, cross calibration, and feasibility studies of space-based instrumentation for the benefit of future high energy missions like Athena, and the best exploitation of existing observatories. AHEAD will support the community via grants for collaborative studies, dissemination of results, and promotion of workshops. A strong public outreach package will ensure that the domain is well publicized at national, European and International level. Networking, joint research activities and access to infrastructures as devised in AHEAD, will serve to establish strong connections between institutes and industry to create the basis for a more rapid advancement of high-energy astrophysical science, space oriented instrumentation and cutting-edge sensor technology in Europe. This enables the development of new technologies and the associated growth of the European technology market with a dedicated technology innovation package, as well as the creation of a new generation of researchers.

  7. In Situ Determination of Structure and Fluctuations of Coexisting Fluid Membrane Domains

    PubMed Central

    Heftberger, Peter; Kollmitzer, Benjamin; Rieder, Alexander A.; Amenitsch, Heinz; Pabst, Georg

    2015-01-01

    Biophysical understanding of membrane domains requires accurate knowledge of their structural details and elasticity. We report on a global small angle x-ray scattering data analysis technique for coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains in fully hydrated multilamellar vesicles. This enabled their detailed analysis for differences in membrane thickness, area per lipid, hydrocarbon chain length, and bending fluctuation as demonstrated for two ternary mixtures (DOPC/DSPC/CHOL and DOPC/DPPC/CHOL) at different cholesterol concentrations. Lo domains were found to be ∼10 Å thicker, and laterally up to 20 Å2/lipid more condensed than Ld domains. Their bending fluctuations were also reduced by ∼65%. Increase of cholesterol concentration caused significant changes in structural properties of Ld, while its influence on Lo properties was marginal. We further observed that temperature-induced melting of Lo domains is associated with a diffusion of cholesterol to Ld domains and controlled by Lo/Ld thickness differences. PMID:25692590

  8. Investigation of system integration methods for bubble domain flight recorders

    NASA Technical Reports Server (NTRS)

    Chen, T. T.; Bohning, O. D.

    1975-01-01

    System integration methods for bubble domain flight records are investigated. Bubble memory module packaging and assembly, the control electronics design and construction, field coils, and permanent magnet bias structure design are studied. A small 60-k bit engineering model was built and tested to demonstrate the feasibility of the bubble recorder. Based on the various studies performed, a projection is made on a 50,000,000-bit prototype recorder. It is estimated that the recorder will occupy 190 cubic in., weigh 12 lb, and consume 12 w power when all of its four tracks are operated in parallel at 150 kHz data rate.

  9. Chain ordering of hybrid lipids can stabilize domains in saturated/hybrid/cholesterol lipid membranes

    NASA Astrophysics Data System (ADS)

    Yamamoto, T.; Brewster, R.; Safran, S. A.

    2010-07-01

    We use a liquid-crystal model to predict that hybrid lipids (lipids that have one saturated and one unsaturated tail) can stabilize line interfaces between domains in mixed membranes of saturated lipids, hybrid lipids, and cholesterol (SHC membranes). The model predicts the phase separation of SHC membranes with both parabolic and loop binodals depending on the cholesterol concentration, modeled via an effective pressure. In some cases, the hybrid lipids can reduce the line tension to zero in SHC membranes at temperatures that approach the critical temperature as the pressure is increased. The differences in the hybrid saturated tail conformational order in bulk and at the interface are responsible for the reduction of the line tension.

  10. Lipid Membrane Deformation Accompanied by Disk-to-Ring Shape Transition of Cholesterol-Rich Domains.

    PubMed

    Ryu, Yong-Sang; Yoo, Daehan; Wittenberg, Nathan J; Jordan, Luke R; Lee, Sin-Doo; Parikh, Atul N; Oh, Sang-Hyun

    2015-07-15

    During vesicle budding or endocytosis, biomembranes undergo a series of lipid- and protein-mediated deformations involving cholesterol-enriched lipid rafts. If lipid rafts of high bending rigidities become confined to the incipient curved membrane topology such as a bud-neck interface, they can be expected to reform as ring-shaped rafts. Here, we report on the observation of a disk-to-ring shape morpho-chemical transition of a model membrane in the absence of geometric constraints. The raft shape transition is triggered by lateral compositional heterogeneity and is accompanied by membrane deformation in the vertical direction, which is detected by height-sensitive fluorescence interference contrast microscopy. Our results suggest that a flat membrane can become curved simply by dynamic changes in local chemical composition and shape transformation of cholesterol-rich domains. PMID:26053547

  11. Multiple membrane-cytoplasmic domain contacts in the cystic fibrosis transmembrane conductance regulator (CFTR) mediate regulation of channel gating.

    PubMed

    He, Lihua; Aleksandrov, Andrei A; Serohijos, Adrian W R; Hegedus, Tamás; Aleksandrov, Luba A; Cui, Liying; Dokholyan, Nikolay V; Riordan, John R

    2008-09-26

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common mutation, deletion of phenylalanine 508 (DeltaF508) and many other disease-associated mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops (CL) of the membrane-spanning domains (MSD). A recently constructed computational model of the CFTR three-dimensional structure, supported by experimental data (Serohijos, A. W., Hegedus, T., Aleksandrov, A. A., He, L., Cui, L., Dokholyan, N. V., and Riordan, J. R. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 3256-3261) revealed that several of these mutations including DeltaF508 disrupted interfaces between these domains. Here we have used cysteine cross-linking experiments to verify all NBD/CL interfaces predicted by the structural model and observed that their cross-linking has a variety of different effects on channel gating. The interdomain contacts comprise aromatic clusters important for stabilization of the interfaces and also involve the Q-loops and X-loops that are in close proximity to the ATP binding sites. Cross-linking of all domain-swapping contacts between NBDs and MSD cytoplasmic loops in opposite halves of the protein rapidly and reversibly arrest single channel gating while those in the same halves have lesser impact. These results reinforce the idea that mediation of regulatory signals between cytoplasmic- and membrane-integrated domains of the CFTR channel apparently relies on an array of precise but highly dynamic interdomain structural joints. PMID:18658148

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

    SciTech Connect

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

    2010-08-13

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

  13. The solution structure of the C-terminal domain of NfeD reveals a novel membrane-anchored OB-fold.

    PubMed

    Kuwahara, Yohta; Ohno, Ayako; Morii, Taichi; Yokoyama, Hideshi; Matsui, Ikuo; Tochio, Hidehito; Shirakawa, Masahiro; Hiroaki, Hidekazu

    2008-11-01

    Nodulation formation efficiency D (NfeD) is a member of a class of membrane-anchored ClpP-class proteases. There is a second class of NfeD homologs that lack the ClpP domain. The genes of both NfeD classes usually are part of an operon that also contains a gene for a prokaryotic homolog of stomatin. (Stomatin is a major integral-membrane protein of mammalian erythrocytes.) Such NfeD/stomatin homolog gene pairs are present in more than 290 bacterial and archaeal genomes, and their protein products may be part of the machinery used for quality control of membrane proteins. Herein, we report the structure of the isolated C-terminal domain of PH0471, a Pyrococcus horikoshii NfeD homolog, which lacks the ClpP domain. This C-terminal domain (termed NfeDC) contains a five-strand beta-barrel, which is structurally very similar to the OB-fold (oligosaccharide/oligonucleotide-binding fold) domain. However, there is little sequence similarity between it and previously characterized OB-fold domains. The NfeDC domain lacks the conserved surface residues that are necessary for the binding of an OB-fold domain to DNA/RNA, an ion. Instead, its surface is composed of residues that are uniquely conserved in NfeD homologs and that form the structurally conserved surface turns and beta-bulges. There is also a conserved tryptophan present on the surface. We propose that, in general, NfeDC domains may interact with other spatially proximal membrane proteins and thereby regulate their activities. PMID:18687870

  14. DNA damage targets PKC{eta} to the nuclear membrane via its C1b domain

    SciTech Connect

    Tamarkin, Ana; Zurgil, Udi; Braiman, Alex; Hai, Naama; Krasnitsky, Ella; Maissel, Adva; Ben-Ari, Assaf; Yankelovich, Liat; Livneh, Etta

    2011-06-10

    Translocation to cellular membranes is one of the hallmarks of PKC activation, occurring as a result of the generation of lipid secondary messengers in target membrane compartments. The activation-induced translocation of PKCs and binding to membranes is largely directed by their regulatory domains. We have previously reported that PKC{eta}, a member of the novel subfamily and an epithelial specific isoform, is localized at the cytoplasm and ER/Golgi and is translocated to the plasma membrane and the nuclear envelope upon short-term activation by PMA. Here we show that PKC{eta} is shuttling between the cytoplasm and the nucleus and that upon etoposide induced DNA damage is tethered at the nuclear envelope. Although PKC{eta} expression and its phosphorylation on the hydrophobic motif (Ser675) are increased by etoposide, this phosphorylation is not required for its accumulation at the nuclear envelope. Moreover, we demonstrate that the C1b domain is sufficient for translocation to the nuclear envelope. We further show that, similar to full-length PKC{eta}, the C1b domain could also confer protection against etoposide-induced cell death. Our studies demonstrate translocation of PKC{eta} to the nuclear envelope, and suggest that its spatial regulation could be important for its cellular functions including effects on cell death.

  15. Thyrotropin-releasing hormone-induced depletion of G(q)alpha/G(11)alpha proteins from detergent-insensitive membrane domains.

    PubMed

    Pesanová, Z; Novotný, J; Cerný, J; Milligan, G; Svoboda, P

    1999-12-24

    The role of detergent-insensitive membrane domains (DIMs) in desensitisation of the G protein-coupled receptor-mediated hormone response was studied in clone E2M11 of HEK293 cells which stably express high levels of both thyrotropin-releasing hormone (TRH) receptors and G(11)alpha G protein. DIMs were prepared by flotation in equilibrium sucrose density gradients and characterised by a panel of membrane markers representing peripheral, glycosylphosphatidylinositol-bound as well as integral membrane proteins (caveolin, CD29, CD55, CD59, CD147, the alpha subunit of Na, K-ATPase) and enzyme activities (alkaline phosphatase, adenylyl cyclase). Caveolin-containing DIMs represented only a small fraction of the overall pool of G(q)alpha/G(11)alpha-rich domains. Prolonged stimulation of E2M11 cells with TRH resulted in dramatic depletion of G(q)alpha/G(11)alpha from all DIMs, which was paralleled by a concomitant G(q)alpha/G(11)alpha increase in the high-density gradient fractions containing the bulk-phase membrane constituents soluble in 1% Triton X-100. Distribution of membrane markers was unchanged under these conditions. Membrane domains thus represent a substantial structural determinant of the G protein pool relevant to desensitisation of hormone action. PMID:10611479

  16. Structure and dynamics of nano-sized raft-like domains on the plasma membrane

    NASA Astrophysics Data System (ADS)

    Herrera, Fernando E.; Pantano, Sergio

    2012-01-01

    Cell membranes are constitutively composed of thousands of different lipidic species, whose specific organization leads to functional heterogeneities. In particular, sphingolipids, cholesterol and some proteins associate among them to form stable nanoscale domains involved in recognition, signaling, membrane trafficking, etc. Atomic-detail information in the nanometer/second scale is still elusive to experimental techniques. In this context, molecular simulations on membrane systems have provided useful insights contributing to bridge this gap. Here we present the results of a series of simulations of biomembranes representing non-raft and raft-like nano-sized domains in order to analyze the particular structural and dynamical properties of these domains. Our results indicate that the smallest (5 nm) raft domains are able to preserve their distinctive structural and dynamical features, such as an increased thickness, higher ordering, lower lateral diffusion, and specific lipid-ion interactions. The insertion of a transmembrane protein helix into non-raft, extended raft-like, and raft-like nanodomain environments result in markedly different protein orientations, highlighting the interplay between the lipid-lipid and lipid-protein interactions.

  17. Allosteric signalling in the outer membrane translocation domain of PapC usher

    PubMed Central

    Farabella, Irene; Pham, Thieng; Henderson, Nadine S; Geibel, Sebastian; Phan, Gilles; Thanassi, David G; Delcour, Anne H; Waksman, Gabriel; Topf, Maya

    2014-01-01

    PapC ushers are outer-membrane proteins enabling assembly and secretion of P pili in uropathogenic E. coli. Their translocation domain is a large β-barrel occluded by a plug domain, which is displaced to allow the translocation of pilus subunits across the membrane. Previous studies suggested that this gating mechanism is controlled by a β-hairpin and an α-helix. To investigate the role of these elements in allosteric signal communication, we developed a method combining evolutionary and molecular dynamics studies of the native translocation domain and mutants lacking the β-hairpin and/or the α-helix. Analysis of a hybrid residue interaction network suggests distinct regions (residue ‘communities’) within the translocation domain (especially around β12–β14) linking these elements, thereby modulating PapC gating. Antibiotic sensitivity and electrophysiology experiments on a set of alanine-substitution mutants confirmed functional roles for four of these communities. This study illuminates the gating mechanism of PapC ushers and its importance in maintaining outer-membrane permeability. DOI: http://dx.doi.org/10.7554/eLife.03532.001 PMID:25271373

  18. Acute Acidification of Stratum Corneum Membrane Domains Using Polyhydroxyl Acids Improves Lipid Processing and Inhibits Degradation of Corneodesmosomes

    PubMed Central

    Hachem, Jean-Pierre; Roelandt, Truus; Schürer, Nanna; Pu, Xu; Fluhr, Joachim; Giddelo, Christina; Man, Mao-Qiang; Crumrine, Debra; Roseeuw, Diane; Feingold, Kenneth R.; Mauro, Theodora; Elias, Peter M.

    2010-01-01

    Neutralization of the normally acidic stratum corneum (SC) has deleterious consequences for permeability barrier homeostasis and SC integrity/cohesion attributable to serine proteases (SPs) activation leading to deactivation/degradation of lipid-processing enzymes and corneodesmosomes (CD). As an elevated pH compromises SC structure and function, we asked here whether SC hyperacidification would improve the structure and function. We lowered the pH of mouse SC using two polyhydroxyl acids (PHA), lactobionic acid (LBA), or gluconolactone (GL). Applications of the PHA reduced the pH at all levels of SC of hairless mouse, with further selective acidification of SC membrane domains, as shown by fluorescence lifetime imaging. Hyperacidification improved permeability barrier homeostasis, attributable to increased activities of two key membrane-localized, ceramide-generating hydrolytic enzymes (β-glucocerebrosidase and acidic sphingomyelinase), which correlated with accelerated extracellular maturation of SC lamellar membranes. Hyperacidification generated “supernormal” SC integrity/cohesion, attributable to an SP-dependent decreased degradation of desmoglein-1 (DSG1) and the induction of DSG3 expression in lower SC. As SC hyperacidification improves the structure and function, even of normal epidermis, these studies lay the groundwork for an assessment of the potential utility of SC acidification as a therapeutic strategy for inflammatory dermatoses, characterized by abnormalities in barrier function, cohesion, and surface pH. PMID:19741713

  19. Acute acidification of stratum corneum membrane domains using polyhydroxyl acids improves lipid processing and inhibits degradation of corneodesmosomes.

    PubMed

    Hachem, Jean-Pierre; Roelandt, Truus; Schürer, Nanna; Pu, Xu; Fluhr, Joachim; Giddelo, Christina; Man, Mao-Qiang; Crumrine, Debra; Roseeuw, Diane; Feingold, Kenneth R; Mauro, Theodora; Elias, Peter M

    2010-02-01

    Neutralization of the normally acidic stratum corneum (SC) has deleterious consequences for permeability barrier homeostasis and SC integrity/cohesion attributable to serine proteases (SPs) activation leading to deactivation/degradation of lipid-processing enzymes and corneodesmosomes (CD). As an elevated pH compromises SC structure and function, we asked here whether SC hyperacidification would improve the structure and function. We lowered the pH of mouse SC using two polyhydroxyl acids (PHA), lactobionic acid (LBA), or gluconolactone (GL). Applications of the PHA reduced the pH at all levels of SC of hairless mouse, with further selective acidification of SC membrane domains, as shown by fluorescence lifetime imaging. Hyperacidification improved permeability barrier homeostasis, attributable to increased activities of two key membrane-localized, ceramide-generating hydrolytic enzymes (beta-glucocerebrosidase and acidic sphingomyelinase), which correlated with accelerated extracellular maturation of SC lamellar membranes. Hyperacidification generated "supernormal" SC integrity/cohesion, attributable to an SP-dependent decreased degradation of desmoglein-1 (DSG1) and the induction of DSG3 expression in lower SC. As SC hyperacidification improves the structure and function, even of normal epidermis, these studies lay the groundwork for an assessment of the potential utility of SC acidification as a therapeutic strategy for inflammatory dermatoses, characterized by abnormalities in barrier function, cohesion, and surface pH. PMID:19741713

  20. Membrane curvature sensing by the C-terminal domain of complexin

    NASA Astrophysics Data System (ADS)

    Snead, David; Wragg, Rachel T.; Dittman, Jeremy S.; Eliezer, David

    2014-09-01

    Complexin functions at presynaptic nerve terminals to inhibit spontaneous SNARE-mediated synaptic vesicle (SV) exocytosis, while enhancing stimulated neurotransmitter release. The C-terminal domain (CTD) of complexin is essential for its inhibitory function and has been implicated in localizing complexin to SVs via direct membrane interactions. Here we show that complexin’s CTD is highly sensitive to membrane curvature, which it senses via tandem motifs, a C-terminal motif containing a mix of bulky hydrophobic and positively charged residues, and an adjacent amphipathic region that can bind membranes in either a disordered or a helical conformation. Helix formation requires membrane packing defects found on highly curved membrane surfaces. Mutations that disrupt helix formation without disrupting membrane binding compromise complexin’s inhibitory function in vivo. Thus, this membrane curvature-dependent conformational transition, combined with curvature-sensitive binding by the adjacent C-terminal motif, constitute a novel mechanism for activating complexin’s inhibitory function on the surface of SVs.

  1. Structure and analysis of FCHo2 F-BAR domain: a dimerizing and membrane recruitment module that effects membrane curvature.

    PubMed

    Henne, William Mike; Kent, Helen M; Ford, Marijn G J; Hegde, Balachandra G; Daumke, Oliver; Butler, P Jonathan G; Mittal, Rohit; Langen, Ralf; Evans, Philip R; McMahon, Harvey T

    2007-07-01

    A spectrum of membrane curvatures exists within cells, and proteins have evolved different modules to detect, create, and maintain these curvatures. Here we present the crystal structure of one such module found within human FCHo2. This F-BAR (extended FCH) module consists of two F-BAR domains, forming an intrinsically curved all-helical antiparallel dimer with a Kd of 2.5 microM. The module binds liposomes via a concave face, deforming them into tubules with variable diameters of up to 130 nm. Pulse EPR studies showed the membrane-bound dimer is the same as the crystal dimer, although the N-terminal helix changed conformation on membrane binding. Mutation of a phenylalanine on this helix partially attenuated narrow tubule formation, and resulted in a gain of curvature sensitivity. This structure shows a distant relationship to curvature-sensing BAR modules, and suggests how similar coiled-coil architectures in the BAR superfamily have evolved to expand the repertoire of membrane-sculpting possibilities. PMID:17540576

  2. The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae.

    PubMed

    Okumura, Yuuya; Nakamura, Tsuyoshi S; Tanaka, Takayuki; Inoue, Ichiro; Suda, Yasuyuki; Takahashi, Tetsuo; Nakanishi, Hideki; Nakamura, Shugo; Gao, Xiao-Dong; Tachikawa, Hiroyuki

    2016-01-01

    Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation

  3. The Dysferlin Domain-Only Protein, Spo73, Is Required for Prospore Membrane Extension in Saccharomyces cerevisiae

    PubMed Central

    Okumura, Yuuya; Nakamura, Tsuyoshi S.; Tanaka, Takayuki; Inoue, Ichiro; Suda, Yasuyuki; Takahashi, Tetsuo; Nakanishi, Hideki; Nakamura, Shugo; Gao, Xiao-Dong

    2015-01-01

    ABSTRACT Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73∆ mutant revealed that it shows defects similar to those of a spo71∆ mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71∆ mutants, spo73∆ mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy. IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall

  4. Factors Influencing Local Membrane Curvature Induction by N-BAR Domains as Revealed by Molecular Dynamics Simulations

    PubMed Central

    Blood, Philip D.; Swenson, Richard D.; Voth, Gregory A.

    2008-01-01

    N-BAR domains are protein modules that bind to and induce curvature in membranes via a charged concave surface and N-terminal amphipathic helices. Recently, molecular dynamics simulations have demonstrated that the N-BAR domain can induce a strong local curvature that matches the curvature of the BAR domain surface facing the bilayer. Here we present further molecular dynamics simulations that examine in greater detail the roles of the concave surface and amphipathic helices in driving local membrane curvature. We find that the strong curvature induction observed in our previous simulations requires the stable presentation of the charged concave surface to the membrane and is not driven by the membrane-embedded amphipathic helices. Nevertheless, without these amphipathic helices embedded in the membrane, the N-BAR domain does not maintain a close association with the bilayer, and fails to drive membrane curvature. Increasing the membrane negative charge through the addition of PIP2 facilitates closer association with the membrane in the absence of embedded helices. At sufficiently high concentrations, amphipathic helices embedded in the membrane drive membrane curvature independently of the BAR domain. PMID:18469070

  5. Comparison of methods for assessing integrity of equine sperm membranes.

    PubMed

    Foster, M L; Love, C C; Varner, D D; Brinsko, S P; Hinrichs, K; Teague, S; Lacaze, K; Blanchard, T L

    2011-07-15

    Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = -3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = -11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes. PMID:21496902

  6. Lipid domains in supported SM-Chol membranes measured by GISANS

    NASA Astrophysics Data System (ADS)

    Zhernenkov, Mikhail; Dubey, Manish; Toperverg, Boris; Majewski, Jaroslaw; Fitzsimmons, Michael

    2011-03-01

    Cell membranes are known to contain regions (called lipid domains, or rafts) described as sphingolipid-cholesterol assemblies which also may contain a subset of membrane proteins. Currently, the main point of discussion is the methodology to study lipid domains and their sizes. We report on Grazing Incidence Small Angle Neutron Scattering (GISANS) measurements of lipid domains in supported sphingomyelin(SM)-cholesterol(Chol) bilayers in a fully aqueous environment. The model bilayers SM:Chol(2:1), SM:Chol(1:2), and a pure SM were deposited using Langmuir-Blodgett/Langmuir-Schaefer technique at a surface pressure of 10 mN/m and measured at 25rC. First measurements revealed short range inhomogeneities of the order of 100 AA in both binary systems. The control measurement of a pure SM bilayer exhibited nearly no GISANS indicating an absence of lipid domains in the SM bilayer. This observation is consistent with the notion that a single component system studied below the liquid-gel transition temperature will not produce lipid domains. Work was supported by DOE-BES.

  7. TMEM115 is an integral membrane protein of the Golgi complex involved in retrograde transport

    PubMed Central

    Ong, Yan Shan; Tran, Ton Hoai Thi; Gounko, Natalia V.; Hong, Wanjin

    2014-01-01

    ABSTRACT Searching and evaluating the Human Protein Atlas for transmembrane proteins enabled us to identify an integral membrane protein, TMEM115, that is enriched in the Golgi complex. Biochemical and cell biological analysis suggested that TMEM115 has four candidate transmembrane domains located in the N-terminal region. Both the N- and C-terminal domains are oriented towards the cytoplasm. Immunofluorescence analysis supports that TMEM115 is enriched in the Golgi cisternae. Functionally, TMEM115 knockdown or overexpression delays Brefeldin-A-induced Golgi-to-ER retrograde transport, phenocopying cells with mutations or silencing of the conserved oligomeric Golgi (COG) complex. Co-immunoprecipitation and in vitro binding experiments reveals that TMEM115 interacts with the COG complex, and might self-interact to form dimers or oligomers. A short region (residues 206–229) immediately to the C-terminal side of the fourth transmembrane domain is both necessary and sufficient for Golgi targeting. Knockdown of TMEM115 also reduces the binding of the lectins peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA), suggesting an altered O-linked glycosylation profile. These results establish that TMEM115 is an integral membrane protein of the Golgi stack regulating Golgi-to-ER retrograde transport and is likely to be part of the machinery of the COG complex. PMID:24806965

  8. Interactions of Pleckstrin Homology Domains with Membranes: Adding Back the Bilayer via High-Throughput Molecular Dynamics.

    PubMed

    Yamamoto, Eiji; Kalli, Antreas C; Yasuoka, Kenji; Sansom, Mark S P

    2016-08-01

    A molecular simulation pipeline for determining the mode of interaction of pleckstrin homology (PH) domains with phosphatidylinositol phosphate (PIP)-containing lipid bilayers is presented. We evaluate our methodology for the GRP1 PH domain via comparison with structural and biophysical data. Coarse-grained simulations yield a 2D density landscape for PH/membrane interactions alongside residue contact profiles. Predictions of the membrane localization and interactions of 13 PH domains reveal canonical, non-canonical, and dual PIP-binding sites on the proteins. Thus, the PH domains associate with the PIP molecules in the membrane via a highly positively charged loop. Some PH domains exhibit modes of interaction with PIP-containing membranes additional to this canonical binding mode. All 13 PH domains cause a degree of local clustering of PIP molecules upon binding to the membrane. This provides a global picture of PH domain interactions with membranes. The high-throughput approach could be extended to other families of peripheral membrane proteins. PMID:27427480

  9. Two photon fluorescence imaging of lipid membrane domains and potentials using advanced fluorescent probes

    NASA Astrophysics Data System (ADS)

    Kilin, Vasyl; Darwich, Zeinab; Richert, Ludovic; Didier, Pascal; Klymchenko, Andrey; Mély, Yves

    2013-02-01

    Biomembranes are ordered and dynamic nanoscale structures critical for cell functions. The biological functions of the membranes strongly depend on their physicochemical properties, such as electrostatics, phase state, viscosity, polarity and hydration. These properties are essential for the membrane structure and the proper folding and function of membrane proteins. To monitor these properties, fluorescence techniques and notably, two-photon microscopy appear highly suited due to their exquisite sensitivity and their capability to operate in complex biological systems, such as living cells and tissues. In this context, we have developed multiparametric environment-sensitive fluorescent probes tailored for precise location in the membrane bilayer. We notably developed probes of the 3-hydroxychromone family, characterized by an excited state intramolecular proton transfer reaction, which generates two tautomeric emissive species with well-separated emission bands. As a consequence, the response of these probes to changes in their environment could be monitored through changes in the ratios of the two bands, as well as through changes in the fluorescence lifetimes. Using two-photon ratiometric imaging and FLIM, these probes were used to monitor the surface membrane potential, and were applied to detect apoptotic cells and image membrane domains.

  10. A new integrated membrane filtration and chromatographic device.

    PubMed

    Xu, Yanke; Sirkar, Kamalesh K; Dai, Xiao-Ping; Luo, Robert G

    2005-01-01

    To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration. PMID:15801803

  11. Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I

    PubMed Central

    Kaila, Ville R. I.; Wikström, Mårten; Hummer, Gerhard

    2014-01-01

    Complex I serves as the primary electron entry point into the mitochondrial and bacterial respiratory chains. It catalyzes the reduction of quinones by electron transfer from NADH, and couples this exergonic reaction to the translocation of protons against an electrochemical proton gradient. The membrane domain of the enzyme extends ∼180 Å from the site of quinone reduction to the most distant proton pathway. To elucidate possible mechanisms of the long-range proton-coupled electron transfer process, we perform large-scale atomistic molecular dynamics simulations of the membrane domain of complex I from Escherichia coli. We observe spontaneous hydration of a putative proton entry channel at the NuoN/K interface, which is sensitive to the protonation state of buried glutamic acid residues. In hybrid quantum mechanics/classical mechanics simulations, we find that the observed water wires support rapid proton transfer from the protein surface to the center of the membrane domain. To explore the functional relevance of the pseudosymmetric inverted-repeat structures of the antiporter-like subunits NuoL/M/N, we constructed a symmetry-related structure of a possible alternate-access state. In molecular dynamics simulations, we find the resulting structural changes to be metastable and reversible at the protein backbone level. However, the increased hydration induced by the conformational change persists, with water molecules establishing enhanced lateral connectivity and pathways for proton transfer between conserved ionizable residues along the center of the membrane domain. Overall, the observed water-gated transitions establish conduits for the unidirectional proton translocation processes, and provide a possible coupling mechanism for the energy transduction in complex I. PMID:24778264

  12. Functional analysis of the transmembrane domain in paramyxovirus F protein-mediated membrane fusion

    PubMed Central

    Bissonnette, Mei Lin Z.; Donald, Jason E.; DeGrado, William F.; Jardetzky, Theodore S.; Lamb, Robert A.

    2009-01-01

    To enter cells enveloped viruses use fusion-mediating glycoproteins to facilitate the merger of the viral and host cell membranes. These glycoproteins undergo large-scale irreversible refolding during membrane fusion. The paramyxovirus parainfluenza virus 5 (PIV5) mediates membrane merger through its fusion protein (F). The transmembrane (TM) domains of viral fusion proteins are typically required for fusion. The TM domain of F is particularly interesting in that it is potentially unusually long; multiple calculations suggest a TM helix length between 25 and 48 residues. Oxidative cross-linking of single cysteine substitutions indicates the F TM trimer forms a helical bundle within the membrane. To assess the functional role of the PIV5 F protein TM domain, alanine scanning mutagenesis was performed. Two residues located in the outer leaflet of the bilayer are critical for fusion. Multiple amino acid substitutions at these positions indicate the physical properties of the side chain play a critical role in supporting or blocking fusion. Analysis of intermediate steps in F protein refolding indicated that the mutants were not trapped at the open stalk intermediate or the prehairpin intermediate. Incorporation of a known F protein destabilizing mutation that causes a hyperfusogenic phenotype restored fusion activity to the mutants. Further, altering the curvature of the lipid bilayer by addition of oleic acid promoted fusion of the F protein mutants. In aggregate, these data indicate that the TM domain plays a functional role in fusion beyond merely anchoring the protein in the viral envelope and that it can affect the structures and steady-state concentrations of the various conformational intermediates en route to the final postfusion state. We suggest that the unusual length of this TM helix might allow it to serve as a template for formation of or specifically stabilize the lipid stalk intermediate in fusion. PMID:19121325

  13. A conserved polybasic domain mediates plasma membrane targeting of Lgl and its regulation by hypoxia

    PubMed Central

    Dong, Wei; Zhang, Xuejing; Liu, Weijie; Chen, Yi-jiun; Huang, Juan; Austin, Erin; Celotto, Alicia M.; Jiang, Wendy Z.; Palladino, Michael J.; Jiang, Yu; Hammond, Gerald R.V.

    2015-01-01

    Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase–mediated phosphorylations. Instead, we identified an evolutionarily conserved polybasic (PB) domain that targets Lgl to the PM via electrostatic binding to membrane phosphatidylinositol phosphates. Such PB domain–mediated PM targeting is inhibited by hypoxia, which reduces inositol phospholipid levels on the PM through adenosine triphosphate depletion. Moreover, Lgl PB domain contains all the identified phosphorylation sites of aPKC and Aurora kinases, providing a molecular mechanism by which phosphorylations neutralize the positive charges on the PB domain to inhibit Lgl PM targeting. PMID:26483556

  14. Integral Membrane Protein Sorting to Vacuoles in Plant Cells: Evidence for Two Pathways

    PubMed Central

    Jiang, Liwen; Rogers, John C.

    1998-01-01

    Plant cells may contain two functionally distinct vacuolar compartments. Membranes of protein storage vacuoles (PSV) are marked by the presence of α-tonoplast intrinsic protein (TIP), whereas lytic vacuoles (LV) are marked by the presence of γ-TIP. Mechanisms for sorting integral membrane proteins to the different vacuoles have not been elucidated. Here we study a chimeric integral membrane reporter protein expressed in tobacco suspension culture protoplasts whose traffic was assessed biochemically by following acquisition of complex Asn-linked glycan modifications and proteolytic processing, and whose intracellular localization was determined with confocal immunofluorescence. We show that the transmembrane domain of the plant vacuolar sorting receptor BP-80 directs the reporter protein via the Golgi to the LV prevacuolar compartment, and attaching the cytoplasmic tail (CT) of γ-TIP did not alter this traffic. In contrast, the α-TIP CT prevented traffic of the reporter protein through the Golgi and caused it to be localized in organelles separate from ER and from Golgi and LV prevacuolar compartment markers. These organelles had a buoyant density consistent with vacuoles, and α-TIP protein colocalized in them with the α-TIP CT reporter protein when the two were expressed together in protoplasts. These results are consistent with two separate pathways to vacuoles for membrane proteins: a direct ER to PSV pathway, and a separate pathway via the Golgi to the LV. PMID:9832548

  15. Lipid packing drives the segregation of transmembrane helices into disordered lipid domains in model membranes

    PubMed Central

    Schäfer, Lars V.; de Jong, Djurre H.; Holt, Andrea; Rzepiela, Andrzej J.; de Vries, Alex H.; Poolman, Bert; Killian, J. Antoinette; Marrink, Siewert J.

    2011-01-01

    Cell membranes are comprised of multicomponent lipid and protein mixtures that exhibit a complex partitioning behavior. Regions of structural and compositional heterogeneity play a major role in the sorting and self-assembly of proteins, and their clustering into higher-order oligomers. Here, we use computer simulations and optical microscopy to study the sorting of transmembrane helices into the liquid-disordered domains of phase-separated model membranes, irrespective of peptide–lipid hydrophobic mismatch. Free energy calculations show that the enthalpic contribution due to the packing of the lipids drives the lateral sorting of the helices. Hydrophobic mismatch regulates the clustering into either small dynamic or large static aggregates. These results reveal important molecular driving forces for the lateral organization and self-assembly of transmembrane helices in heterogeneous model membranes, with implications for the formation of functional protein complexes in real cells. PMID:21205902

  16. Investigation of domain size in polymer membranes using double quantum filtered spin diffusion MAS NMR.

    SciTech Connect

    Fujimoto, Cy H.; Alam, Todd Michael; Cherry, Brian Ray; Cornelius, Christopher James

    2005-02-01

    Solid-state {sup 1}H magic angle spinning (MAS) NMR was used to investigate sulfonated Diels-Alder poly(phenlylene) polymer membranes. Under high spinning speed {sup 1}H MAS conditions, the proton environments of the sulfonic acid and phenylene polymer backbone are resolved. A double-quantum (DQ) filter using the rotor-synchronized back-to-back (BABA) NMR multiple-pulse sequence allowed the selective suppression of the sulfonic proton environment in the {sup 1}H MAS NMR spectra. This DQ filter in conjunction with a spin diffusion NMR experiment was then used to measure the domain size of the sulfonic acid component within the membrane. In addition, the temperature dependence of the sulfonic acid spin-spin relaxation time (T{sub 2}) was determined, providing an estimate of the activation energy for the proton dynamics of the dehydrated membrane.

  17. High resolution nanomechanical characterization of multi-domain model membranes by fast Force Volume.

    PubMed

    Seghezza, Silvia; Dante, Silvia; Diaspro, Alberto; Canale, Claudio

    2015-12-01

    Plasma membrane is a complex structure, mainly composed by lipids and proteins, which plays a pivotal role in cell metabolism by regulating its selective permeability to ions and molecules. According to the "raft hypothesis", lipids in the bilayer are not forming a structurally passive solvent, but are rather organized in specific domains, which present different structural and functional characteristics. The mechanical properties of the lipid part of plasma membrane have been recently characterized through Atomic Force Microscopy, by analyzing the features of force vs distance curves collected on supported lipid bilayers (SLBs). In case of lipid domains sizing from tens to hundreds of nanometers, which mimic in a good way the lateral organization of real membranes, a high lateral resolution and a large number of curves are often required for properly expressing the complexity of the system, with a consequent exponential growth of acquisition and processing time. In this paper we propose a method, based on a recently developed high speed Force Volume technique and on home-built data processing software, for the mechanical characterization of nanostructured SLBs. With our software we have been able to process data set composed by tens of thousands of curves, collected with a spatial resolution ranging from 8 to 40 nm/pixel. Multiparametric maps and distribution histograms produced by our analysis allowed identifying a specific behavior for each lipid phase in the investigated model membranes, even in presence of nanosized features. PMID:26224416

  18. Membrane insertion of the Bacillus thuringiensis Cry1Ab toxin: single mutation in domain II block partitioning of the toxin into the brush border membrane.

    PubMed

    Nair, Manoj S; Liu, Xinyan Sylvia; Dean, Donald H

    2008-05-27

    The umbrella and penknife models hypothesize that insecticidal Bacillus thuringiensis Cry toxins partition into the apical membrane of the insect midgut by insertion of only two alpha-helices from domain I of the protein, alpha-helices 4 and 5 in the case of the umbrella model and alpha-helices 5 and 6 in the case of the penknife model. Neither model envisages membrane partitioning by domains II and III. In this study, we present data suggesting that mutations in the domain II residue, F371, affect insertion of the whole toxin into Manduca sexta brush border membrane vesicles (BBMVs). Using steady state fluorescence measurements combined with a proteinase K protection assay, we show that mutants of F371 have lost their ability to insert into the BBMV, even though binding to cadherin is almost unaffected. The study also identifies a difference in partitioning of toxins into artificial lipid vesicles (SUVs) as opposed to native BBMVs. While the F371 mutations block insertion of domains I and II into BBMVs, they only block domain II insertion into SUVs. Bioassay and voltage clamping of midguts also confirm the fluorescence data that the noninserting mutants are nontoxic. Our study leads us to propose that, in contrast to previous models of individual free helices inserting into the membrane, the toxin enters into the membrane as a whole molecule or oligomers of the molecule, wherein the domain II residue F371 has a vital role to play in membrane insertion. PMID:18457427

  19. Understanding the Role of Amphipathic Helices in N-BAR Domain Driven Membrane Remodeling

    PubMed Central

    Cui, Haosheng; Mim, Carsten; Vázquez, Francisco X.; Lyman, Edward; Unger, Vinzenz M.; Voth, Gregory A.

    2013-01-01

    Endophilin N-BAR (N-terminal helix and Bin/amphiphysin/Rvs) domain tubulates and vesiculates lipid membranes in vitro via its crescent-shaped dimer and four amphipathic helices that penetrate into membranes as wedges. Like F-BAR domains, endophilin N-BAR also forms a scaffold on membrane tubes. Unlike F-BARs, endophilin N-BARs have N-terminal H0 amphipathic helices that are proposed to interact with other N-BARs in oligomer lattices. Recent cryo-electron microscopy reconstructions shed light on the organization of the N-BAR lattice coats on a nanometer scale. However, because of the resolution of the reconstructions, the precise positioning of the amphipathic helices is still ambiguous. In this work, we applied a coarse-grained model to study various membrane remodeling scenarios induced by endophilin N-BARs. We found that H0 helices of N-BARs prefer to align in an antiparallel manner at two ends of the protein to form a stable lattice. The deletion of H0 helices causes disruption of the lattice. In addition, we analyzed the persistence lengths of the protein-coated tubes and found that the stiffness of endophilin N-BAR-coated tubules qualitatively agrees with previous experimental work studying N-BAR-coated tubules. Large-scale simulations on membrane liposomes revealed a systematic relation between H0 helix density and local membrane curvature fluctuations. The data also suggest that the H0 helix is required for BARs to form organized structures on the liposome, further illustrating its important function. PMID:23442862

  20. Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3.

    PubMed

    Wang, Huayi; Sun, Liming; Su, Lijing; Rizo, Josep; Liu, Lei; Wang, Li-Feng; Wang, Fu-Sheng; Wang, Xiaodong

    2014-04-10

    Programmed necrotic cell death induced by the tumor necrosis factor alpha (TNF-α) family of cytokines is dependent on a kinase cascade consisting of receptor-interacting kinases RIP1 and RIP3. How these kinase activities cause cells to die by necrosis is not known. The mixed lineage kinase domain-like protein MLKL is a functional RIP3 substrate that binds to RIP3 through its kinase-like domain but lacks kinase activity of its own. RIP3 phosphorylates MLKL at the T357 and S358 sites. Reported here is the development of a monoclonal antibody that specifically recognizes phosphorylated MLKL in cells dying of this pathway and in human liver biopsy samples from patients suffering from drug-induced liver injury. The phosphorylated MLKL forms an oligomer that binds to phosphatidylinositol lipids and cardiolipin. This property allows MLKL to move from the cytosol to the plasma and intracellular membranes, where it directly disrupts membrane integrity, resulting in necrotic death. PMID:24703947

  1. Formation of irreversibly bound annexin A1 protein domains on POPC/POPS solid supported membranes.

    PubMed

    Faiss, Simon; Kastl, Katja; Janshoff, Andreas; Steinem, Claudia

    2008-01-01

    The specific interaction of annexin A1 with phospholipid bilayers is scrutinized by means of scanning force and fluorescence microscopy, quartz crystal microbalance, ellipsometry, and modeled by dynamic Monte Carlo simulations. It was found that POPC/POPS bilayers exhibit phase separation in POPC- and POPS-enriched domains as a function of Ca2+ concentration. Annexin A1 interacts with POPC/POPS bilayers by forming irreversibly bound protein domains with monolayer thickness on POPS-enriched nanodomains, while the attachment of proteins to the POPC-enriched regions is fully reversible. A thorough kinetic analysis of the process reveals that both, the binding constant of annexin A1 at the POPC-rich areas as well as the irreversible adsorption rate to the POPS-rich domains increases with calcium ion concentration. Based on the thermodynamic and kinetic data, a possible mechanism of the annexin A1 membrane interaction can be proposed. PMID:18237543

  2. Integral ceramic superstructure evaluation using time domain optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Sinescu, Cosmin; Bradu, Adrian; Topala, Florin I.; Negrutiu, Meda Lavinia; Duma, Virgil-Florin; Podoleanu, Adrian G.

    2014-02-01

    Optical Coherence Tomography (OCT) is a non-invasive low coherence interferometry technique that includes several technologies (and the corresponding devices and components), such as illumination and detection, interferometry, scanning, adaptive optics, microscopy and endoscopy. From its large area of applications, we consider in this paper a critical aspect in dentistry - to be investigated with a Time Domain (TD) OCT system. The clinical situation of an edentulous mandible is considered; it can be solved by inserting 2 to 6 implants. On these implants a mesostructure will be manufactured and on it a superstructure is needed. This superstructure can be integral ceramic; in this case materials defects could be trapped inside the ceramic layers and those defects could lead to fractures of the entire superstructure. In this paper we demonstrate that a TD-OCT imaging system has the potential to properly evaluate the presence of the defects inside the ceramic layers and those defects can be fixed before inserting the prosthesis inside the oral cavity. Three integral ceramic superstructures were developed by using a CAD/CAM technology. After the milling, the ceramic layers were applied on the core. All the three samples were evaluated by a TD-OCT system working at 1300 nm. For two of the superstructures evaluated, no defects were found in the most stressed areas. The third superstructure presented four ceramic defects in the mentioned areas. Because of those defects the superstructure may fracture. The integral ceramic prosthesis was send back to the dental laboratory to fix the problems related to the material defects found. Thus, TD-OCT proved to be a valuable method for diagnosing the ceramic defects inside the integral ceramic superstructures in order to prevent fractures at this level.

  3. Nonspecific Binding Domains in Lipid Membranes Induced by Phospholipase A2.

    PubMed

    Hong, Chia Yee; Han, Chung-Ta; Chao, Ling

    2016-07-12

    Phospholipase A2 (PLA2) is a peripheral membrane protein that can hydrolyze phospholipids to produce lysolipids and fatty acids. It has been found to play crucial roles in various cellular processes and is thought as a potential candidate for triggering drug release from liposomes for medical treatment. Here, we directly observed that PLA2 hydrolysis reaction can induce the formation of PLA2-binding domains at lipid bilayer interface and found that the formation was significantly influenced by the fluidity of the lipid bilayer. We prepared supported lipid bilayers (SLBs) with various molar ratios of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to adjust the reactivity and fluidity of the lipid bilayers. A significant amount of the PLA2-induced domains was observed in mixtures of DPPC and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) but not in either pure DPPC or pure DOPC bilayer, which might be the reason that previous studies rarely observed these domains in lipid bilayer systems. The fluorescently labeled PLA2 experiment showed that newly formed domains acted as binding templates for PLA2. The AFM result showed that the induced domain has stepwise plateau structure, suggesting that PLA2 hydrolysis products may align as bilayers and accumulate layer by layer on the support, and the hydrophobic acyl chains at the side of the layer structure may be exposed to the outside aqueous environment. The introduced hydrophobic region could have hydrophobic interactions with proteins and therefore can attract the binding of not only PLA2 but also other types of proteins such as proteoglycans and streptavidin. The results suggest that the formation of PLA2-induced domains may convert part of a zwitterionic nonsticky lipid membrane to a site where biomolecules can nonspecifically bind. PMID:27218880

  4. Crystal Structures of the Outer Membrane Domain of Intimin and Invasin from Enterohemorrhagic E. coli and Enteropathogenic Y. pseudotuberculosis

    SciTech Connect

    Fairman, James W.; Dautin, Nathalie; Wojtowicz, Damian; Liu, Wei; Noinaj, Nicholas; Barnard, Travis J.; Udho, Eshwar; Przytycka, Teresa M.; Cherezov, Vadim; Buchanan, Susan K.

    2012-12-10

    Intimins and invasins are virulence factors produced by pathogenic Gram-negative bacteria. They contain C-terminal extracellular passenger domains that are involved in adhesion to host cells and N-terminal {beta} domains that are embedded in the outer membrane. Here, we identify the domain boundaries of an E. coli intimin {beta} domain and use this information to solve its structure and the {beta} domain structure of a Y. pseudotuberculosis invasin. Both {beta} domain structures crystallized as monomers and reveal that the previous range of residues assigned to the {beta} domain also includes a protease-resistant domain that is part of the passenger. Additionally, we identify 146 nonredundant representative members of the intimin/invasin family based on the boundaries of the highly conserved intimin and invasin {beta} domains. We then use this set of sequences along with our structural data to find and map the evolutionarily constrained residues within the {beta} domain.

  5. Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase.

    PubMed

    Van Horn, Wade D; Kim, Hak-Jun; Ellis, Charles D; Hadziselimovic, Arina; Sulistijo, Endah S; Karra, Murthy D; Tian, Changlin; Sönnichsen, Frank D; Sanders, Charles R

    2009-06-26

    Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK's lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis. PMID:19556511

  6. Structural Plasticity in the Topology of the Membrane-Interacting Domain of HIV-1 gp41

    PubMed Central

    Kyrychenko, Alexander; Freites, J. Alfredo; He, Jing; Tobias, Douglas J.; Wimley, William C.; Ladokhin, Alexey S.

    2014-01-01

    We use a number of computational and experimental approaches to investigate the membrane topology of the membrane-interacting C-terminal domain of the HIV-1 gp41 fusion protein. Several putative transmembrane regions are identified using hydrophobicity analysis based on the Wimley-White scales, including the membrane-proximal external region (MPER). The MPER region is an important target for neutralizing anti-HIV monoclonal antibodies and is believed to have an interfacial topology in the membrane. To assess the possibility of a transmembrane topology of MPER, we examined the membrane interactions of a peptide corresponding to a 22-residue stretch of the MPER sequence (residues 662–683) using fluorescence spectroscopy and oriented circular dichroism. In addition to the previously reported interfacial location, we identify a stable transmembrane conformation of the peptide in synthetic lipid bilayers. All-atom molecular dynamics simulations of the MPER-derived peptide in a lipid bilayer demonstrate a stable helical structure with an average tilt of 24 degrees, with the five tryptophan residues sampling different environments inside the hydrocarbon core of the lipid bilayer, consistent with the observed spectral properties of intrinsic fluorescence. The degree of lipid bilayer penetration obtained by computer simulation was verified using depth-dependent fluorescence quenching of a selectively attached fluorescence probe. Overall, our data indicate that the MPER sequence can have at least two stable conformations in the lipid bilayer, interfacial and transmembrane, and suggest a possibility that external perturbations can switch the topology during physiological functioning. PMID:24507601

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

    PubMed Central

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

    2015-01-01

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

  8. Spectral domain ocular coherence tomography findings pre- and post vitrectomy with fibrovascular membrane delamination for proliferative diabetic retinopathy.

    PubMed

    Dooley, I; Laviers, H; Papavasileiou, E; Mckechnie, C; Zambarakji, H

    2016-01-01

    PurposeTo describe the intraretinal microstructure using serial spectral domain optical coherence tomography (SD-OCT) preceding and following pars plana vitrectomy and delamination of fibrovascular membranes in patients with proliferative diabetic retinopathy (PDR).MethodsThis retrospective, interventional case series includes 28 eyes. Outcome measures included LogMAR distance best-corrected visual acuity (BCVA), SD-OCT integrity of photoreceptor inner and outer segments junction (IS/OS), and integrity of external limiting membrane (ELM).ResultsPre-operative central macular thickness (CMT) was significantly correlated with the final post-operative LogMAR BCVA (Pearson's coefficient r=0.89; P=0.001). The eyes were categorised into three groups based on post-operative IS/OS integrity (group 0: IS/OS intact; group 1: IS/OS irregular but not completely disrupted; group 2: IS/OS completely disrupted). Mean BCVA improved significantly in group 0 (n=9) from 1.13±0.75 preoperatively to 0.34±0.21 (Student's t-test: P=0.06), in group 1 (n=10) the BCVA improved from 0.88±0.56 to 0.58±0.31 (Student's t-test: P=0.053) and in group 2 (n=9) the BCVA improved from 1.64±0.53 to 1.53±0.75 (Student's t-test: P=0.652).IS/OS integrity and ELM integrity at 3 months post operatively, were significantly and positively correlated with final BCVA (Pearson's coefficient: r=0.83, P<0.001 and r=0.72, P<0.001, respectively).ConclusionsPre-operative CMT and post-operative disruption of the IS/OS and ELM are useful prognostic indicators in fibrovascular delamination surgery for patients with PDR. PMID:26403326

  9. Formation and Properties of Membrane-Ordered Domains by Phytoceramide: Role of Sphingoid Base Hydroxylation.

    PubMed

    Marquês, Joaquim T; Cordeiro, André M; Viana, Ana S; Herrmann, Andreas; Marinho, H Susana; de Almeida, Rodrigo F M

    2015-09-01

    Phytoceramide is the backbone of major sphingolipids in fungi and plants and is essential in several tissues of animal organisms, such as human skin. Its sphingoid base, phytosphingosine, differs from that usually found in mammals by the addition of a hydroxyl group to the 4-ene, which may be a crucial factor for the different properties of membrane microdomains among those organisms and tissues. Recently, sphingolipid hydroxylation in animal cells emerged as a key feature in several physiopathological processes. Hence, the study of the biophysical properties of phytosphingolipids is also relevant in that context since it helps us to understand the effects of sphingolipid hydroxylation. In this work, binary mixtures of N-stearoyl-phytoceramide (PhyCer) with palmitoyloleoylphosphatidylcholine (POPC) were studied. Steady-state and time-resolved fluorescence of membrane probes, X-ray diffraction, atomic force microscopy, and confocal microscopy were employed. As for other saturated ceramides, highly rigid gel domains start to form with just ∼5 mol % PhyCer at 24 °C. However, PhyCer gel-enriched domains in coexistence with POPC-enriched fluid present additional complexity since their properties (maximal order, shape, and thickness) change at specific POPC/PhyCer molar ratios, suggesting the formation of highly stable stoichiometric complexes with their own properties, distinct from both POPC and PhyCer. A POPC/PhyCer binary phase diagram, supported by the different experimental approaches employed, is proposed with complexes of 3:1 and 1:2 stoichiometries which are stable at least from ∼15 to ∼55 °C. Thus, it provides mechanisms for the in vivo formation of sphingolipid-enriched gel domains that may account for stable membrane compartments and diffusion barriers in eukaryotic cell membranes. PMID:26262576

  10. Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore

    PubMed Central

    Ohba, Tomoyuki; Schirmer, Eric C.; Nishimoto, Takeharu; Gerace, Larry

    2004-01-01

    Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex. PMID:15611332

  11. Membrane anchoring of diacylglycerol-lactones substituted with rigid hydrophobic acyl domains correlates with biological activities

    PubMed Central

    Raifman, Or; Kolusheva, Sofiya; Comin, Maria J.; Kedei, Noemi; Lewin, Nancy E.; Blumberg, Peter M.; Marquez, Victor E.; Jelinek, Raz

    2009-01-01

    Summary Synthetic diacylglycerol lactones (DAG-lactones) are effective modulators of critical cellular signaling pathways, downstream of the lipophilic second messenger diacylglycerol, that activate a host of protein kinase C (PKC) isozymes as well as other non-kinase proteins that share with PKC similar C1 membrane-targeting domains. A fundamental determinant of the biological activity of these amphiphilic molecules is the nature of their interactions with cellular membranes. This study characterizes the membrane interactions and bilayer anchoring of a series of DAG-lactones in which the hydrophobic moiety is a “molecular rod”, namely a rigid 4-[2-(R-phenyl)ethynyl]benzoate moiety in the acyl position. Application of assays employing chromatic biomimetic vesicles and biophysical techniques reveals that the mode of membrane anchoring of the DAG-lactone derivatives was markedly affected by the presence of the hydrophobic diphenyl rod and by the size of the functional unit displayed at the terminus of the rod. Two primary mechanisms of interaction were observed: surface binding of the DAG-lactones at the lipid/water interface and deep insertion of the ligands into the alkyl core of the lipid bilayer. These membrane-insertion properties could explain the different patterns of PKC translocation from cytosol to membranes induced by the molecular-rod DAG-lactones. This investigation emphasizes that the side-residues of DAG-lactones, rather than simply conferring hydrophobicity, profoundly influence membrane interactions and in that fashion may further contribute to the diversity of biological actions of these synthetic biomimetic ligands. PMID:19961537

  12. 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

    SciTech Connect

    Liu, Zhuo; Lin, Lin; Yuan, Cai; Nicolaes, Gerry A.F.; Chen, Liqing; Meehan, Edward J.; Furie, Bruce; Furie, Barbara; Huang, Mingdong

    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.

  13. Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins.

    PubMed

    Brandt, M E; Riley, B S; Radolf, J D; Norgard, M V

    1990-04-01

    The pathogenic spirochete Borrelia burgdorferi contains a set of integral membrane proteins which were selectively extracted into the detergent phase upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114. Virtually all of these hydrophobic proteins were recognized by antibodies in pooled sera from patients with chronic Lyme arthritis, demonstrating that proteins partitioning into the detergent phase of Triton X-114 encompass the major B. burgdorferi immunogens. Furthermore, most of these immunogenic proteins, including the previously characterized OspA and OspB membrane antigens, could be biosynthetically labeled when B. burgdorferi was incubated in vitro with [3H]palmitate. The OspA and OspB antigens were radioimmunoprecipitated from [3H]palmitate-labeled detergent-phase proteins with monoclonal antibodies, and [3H]palmitate was recovered unaltered from these proteins after sequential alkaline and acid hydrolyses. The combined results provide formal confirmation that the major B. burgdorferi immunogens extracted by Triton X-114 are lipoproteins. The demonstration that B. burgdorferi integral membrane antigens are lipoproteins may explain the basis of their immunogenicity and may help to improve our understanding of the surface topology of B. burgdorferi. PMID:2318538

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

    PubMed Central

    Buyan, Amanda; Kalli, Antreas C.; Sansom, Mark S. P.

    2016-01-01

    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. PMID:27459095

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

    SciTech Connect

    Armstrong, Clare L; Barrett, M; Toppozini, L; Yamani, Zahra; Kucerka, Norbert; Katsaras, John; Fragneto, Giovanna; Rheinstadter, Maikel C

    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.

  16. Isolation and characterization of the outer membrane of Escherichia coli with autodisplayed Z-domains.

    PubMed

    Park, Min; Yoo, Gu; Bong, Ji-Hong; Jose, Joachim; Kang, Min-Jung; Pyun, Jae-Chul

    2015-03-01

    "Autodisplay technology" is an expression technique used to display the various recombinant proteins on the outer membrane (OM) of Escherichia coli. The resulting autodisplayed Z-domain has been used to improve the sensitivity of immunoassays. In this work, a facile isolation method of the OM fraction of E. coli with autodisplayed Z-domains was presented using (1) an enzyme reaction for the hydrolysis of the peptidoglycan layer and (2) short centrifugation steps. The purity of the isolated OM fraction was analyzed. For the estimation of contamination with bacterial proteins from other parts of E. coli, Western blots of marker proteins for the OM (OmpA), periplasm (β-lactamase), inner membrane (SecA), and cytoplasm (β-galactosidase) were performed. Additionally, assays of marker components or enzymes from each part of E. coli were carried out including the OM (KDO), inner membrane (NADH oxidase), periplasm (β-lactamase), and cytoplasm (β-galactosidase). The yield of OM isolation using this new method was determined to be 80% of the total OM amount, with less than 1% being contaminants from other parts of E. coli. PMID:25528472

  17. Neuronal membranes are key to the pathogenesis of Alzheimer's disease: the role of both raft and non-raft membrane domains.

    PubMed

    Williamson, R; Sutherland, C

    2011-03-01

    Membrane rafts are sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Membrane rafts isolated from post-mortem AD brain are enriched in both β-amyloid and phosphorylated tau. Proteolytic processing of APP to generate β-amyloid, the principle component of amyloid plaques, can occur in membrane rafts, implicating them in the pathogenesis of Alzheimer's disease (AD). Secondary to their role in β-amyloid generation, membrane rafts have more recently been implicated in the accumulation, aggregation and degradation of β-amyloid, with evidence supporting a specific role for membrane raft gangliosides in the binding and aggregation of β-amyloid. In addition, membrane domain composition has a direct impact on both the generation of β-amyloid and its subsequent toxic actions and as such is a key target for the development of therapeutic strategies. This mini-review will focus on recent advances in our understanding of the relevance of membrane composition, of both raft and non-raft domains, to AD progression in models and in human disease. We will discuss how manipulation of membrane composition can alter both the proteolytic processing of APP and the subsequent binding and aggregation of β-amyloid peptide. PMID:21222605

  18. The dynamin-binding domains of Dap160/intersectin affect bulk membrane retrieval in synapses

    PubMed Central

    Winther, Åsa M. E.; Jiao, Wei; Vorontsova, Olga; Rees, Kathryn A.; Koh, Tong-Wey; Sopova, Elena; Schulze, Karen L.; Bellen, Hugo J.; Shupliakov, Oleg

    2013-01-01

    Summary Dynamin-associated protein 160 kDa (Dap160)/intersectin interacts with several synaptic proteins and affects endocytosis and synapse development. The functional role of the different protein interaction domains is not well understood. Here we show that Drosophila Dap160 lacking the dynamin-binding SH3 domains does not affect the development of the neuromuscular junction but plays a key role in synaptic vesicle recycling. dap160 mutants lacking dynamin-interacting domains no longer accumulate dynamin properly at the periactive zone, and it becomes dispersed in the bouton during stimulation. This is accompanied by a reduction in uptake of the dye FM1-43 and an accumulation of large vesicles and membrane invaginations. However, we do not observe an increase in the number of clathrin-coated intermediates. We also note a depression in evoked excitatory junction potentials (EJPs) during high-rate stimulation, accompanied by aberrantly large miniature EJPs. The data reveal the important role of Dap160 in the targeting of dynamin to the periactive zone, where it is required to suppress bulk synaptic vesicle membrane retrieval during high-frequency activity. PMID:23321638

  19. Membrane fusion by the GTPase atlastin requires a conserved C-terminal cytoplasmic tail and dimerization through the middle domain

    PubMed Central

    Moss, Tyler J.; Andreazza, Camilla; Verma, Avani; Daga, Andrea; McNew, James A.

    2011-01-01

    The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin in adjacent membranes must associate to bring the ER membranes into molecular contact. Drosophila atlastin dimerizes in the presence of GTPγS but is monomeric with GDP or without nucleotide. Oligomerization requires the juxtamembrane middle domain three-helix bundle, as does efficient GTPase activity. A soluble version of the N-terminal cytoplasmic domain that contains the GTPase domain and the middle domain three-helix bundle serves as a potent, concentration-dependent inhibitor of membrane fusion both in vitro and in vivo. However, atlastin domains lacking the middle domain are without effect. GTP-dependent dimerization of atlastin generates an enzymatically active protein that drives membrane fusion after nucleotide hydrolysis and conformational reorganization. PMID:21690399

  20. An equivalent domain integral for analysis of two-dimensional mixed mode problems

    NASA Technical Reports Server (NTRS)

    Raju, I. S.; Shivakumar, K. N.

    1989-01-01

    An equivalent domain integral (EDI) method for calculating J-integrals for two-dimensional cracked elastic bodies subjected to mixed mode loading is presented. The total and product integrals consist of the sum of an area or domain integral and line integrals on the crack faces. The EDI method gave accurate values of the J-integrals for two mode I and two mixed mode problems. Numerical studies showed that domains consisting of one layer of elements are sufficient to obtain accurate J-integral values. Two procedures for separating the individual modes from the domain integrals are presented. The procedure that uses the symmetric and antisymmetric components of the stress and displacement fields to calculate the individual modes gave accurate values of the integrals for all the problems analyzed.

  1. A Link Between Integral Membrane Protein Expression and Simulated Integration Efficiency

    PubMed Central

    Müller, Axel; Tiemann, Katrin; Saladi, Shyam M.; Galimidi, Rachel P.; Zhang, Bin; Clemons, William M.; Miller, Thomas F.

    2016-01-01

    Integral membrane proteins (IMP) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels widely vary and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design. PMID:27524616

  2. A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency.

    PubMed

    Marshall, Stephen S; Niesen, Michiel J M; Müller, Axel; Tiemann, Katrin; Saladi, Shyam M; Galimidi, Rachel P; Zhang, Bin; Clemons, William M; Miller, Thomas F

    2016-08-23

    Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the experimentally observed expression levels. Demonstration of these trends in both Escherichia coli and Mycobacterium smegmatis suggests that the results are general to other expression systems. This work suggests that IMP integration is a determinant for successful expression, raising the possibility of controlling IMP expression via rational design. PMID:27524616

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

    SciTech Connect

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

    2006-09-01

    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{sup +} 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.

  4. Localization of cell surface glycoproteins in membrane domains associated with the underlying filament network.

    PubMed

    Roos, E; Spiele, H; Feltkamp, C A; Huisman, H; Wiegant, F A; Traas, J; Mesland, D A

    1985-11-01

    To visualize the localization of cell surface constituents in relation to the plasma membrane-associated filament network, we developed a method based on a combination of immunogold labeling and dry-cleaving. For labeling we used trinitrophenyl-derivatized ligand, anti-TNP antibodies, and protein A-coated colloidal gold. Dry-cleaving (Mesland, D. A. M., H. Spiele, and E. Roos, 1981, Exp. Cell Res., 132: 169-184) involves cleavage of lightly fixed critical point-dried cells by means of adhesive tape. Since cells cleave close to the cell surface, the remaining layer is thin enough to be examined in transmission electron microscopy. Using this method, we studied concanavalin A-binding constituents on the medium-facing surface of H35 hepatoma cells. The distribution of the gold particles, which was partly dispersed and partly patchy, coincided strikingly with membrane-associated filaments, and label was virtually absent from areas overlying openings in the filament network. In stereo pairs we observed the label to be localized to areas of somewhat enhanced electron density at the plane of the membrane. These areas were interconnected in a pattern congruent with the filament network. Preliminary observations on wheat germ agglutinin receptors on the hepatoma cells as well as concanavalin A receptors on isolated hepatocytes yielded comparable results. It thus appears that surface glycoproteins, although seemingly randomly distributed as observed in thin sections, may actually be localized to particular membrane domains associated with underlying filaments. PMID:3902855

  5. Transmembrane domain-dependent sorting of proteins to the ER and plasma membrane in yeast.

    PubMed Central

    Rayner, J C; Pelham, H R

    1997-01-01

    Sorting of membrane proteins between compartments of the secretory pathway is mediated in part by their transmembrane domains (TMDs). In animal cells, TMD length is a major factor in Golgi retention. In yeast, the role of TMD signals is less clear; it has been proposed that membrane proteins travel by default to the vacuole, and are prevented from doing so by cytoplasmic signals. We have investigated the targeting of the yeast endoplasmic reticulum (ER) t-SNARE Ufe1p. We show that the amino acid sequence of the Ufe1p TMD is important for both function and ER targeting, and that the requirements for each are distinct. Targeting is independent of Rer1p, the only candidate sorting receptor for TMD sequences currently known. Lengthening the Ufe1p TMD allows transport along the secretory pathway to the vacuole or plasma membrane. The choice between these destinations is determined by the length and composition of the TMD, but not by its precise sequence. A longer TMD is required to reach the plasma membrane in yeast than in animal cells, and shorter TMDs direct proteins to the vacuole. TMD-based sorting is therefore a general feature of the yeast secretory pathway, but occurs by different mechanisms at different points. PMID:9155009

  6. Lipodisks integrated with weak affinity chromatography enable fragment screening of integral membrane proteins.

    PubMed

    Duong-Thi, Minh-Dao; Bergström, Maria; Edwards, Katarina; Eriksson, Jonny; Ohlson, Sten; Ying, Janet To Yiu; Torres, Jaume; Hernández, Víctor Agmo

    2016-02-01

    Membrane proteins constitute the largest class of drug targets but they present many challenges in drug discovery. Importantly, the discovery of potential drug candidates is hampered by the limited availability of efficient methods for screening drug-protein interactions. In this work we present a novel strategy for rapid identification of molecules capable of binding to a selected membrane protein. An integral membrane protein (human aquaporin-1) was incorporated into planar lipid bilayer disks (lipodisks), which were subsequently covalently coupled to porous derivatized silica and packed into HPLC columns. The obtained affinity columns were used in a typical protocol for fragment screening by weak affinity chromatography (WAC), in which one hit was identified out of a 200 compound collection. The lipodisk-based strategy, which ensures a stable and native-like lipid environment for the protein, is expected to work also with other membrane proteins and screening procedures. PMID:26673836

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

    SciTech Connect

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

    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.

  8. Cryogenic Integrated Offset Compensation for Time Domain SQUID Multiplexing

    NASA Astrophysics Data System (ADS)

    Prêle, D.; Voisin, F.; Martino, J.; Bréelle, E.; Bordier, G.; Piat, M.

    2012-06-01

    Superconducting QUantum Interference Device (SQUID) multiplexing is a common technique in the use of large arrays of Transition Edge Sensors (TES). A Time Domain Multiplexer (TDM) combines input TES signals into one output signal using several SQUIDs. Different TES, SQUID and amplifier characteristics induce unavoidable different offsets on the multiplexed signal. Additionally, given the periodicity of the SQUID characteristic, the Flux Locked Loop (FLL) operating point is only defined modulo Φ 0. This can lead to a large output offset. In multiplexed mode, the difference between offsets associated with different pixels can induce a parasitic signal which is often larger than that of the TES. These offset signals drastically constrain the readout dynamic range and thus the maximum gain allowed. They also limit the signal-to-noise ratio, the FLL stability and the multiplexing frequency. Offsets in SQUID readout are discussed and offset compensation for TDM is presented. The dynamic calibration and compensation on a simplified 4:1 TDM are demonstrated in simulation. Dynamic offset compensation is being implemented on a cryogenic SiGe integrated circuit operated at 4 K for 128:1 TDM.

  9. Perspective taking in language: integrating the spatial and action domains

    PubMed Central

    Beveridge, Madeleine E. L.; Pickering, Martin J.

    2013-01-01

    Language is an inherently social behavior. In this paper, we bring together two research areas that typically occupy distinct sections of the literature: perspective taking in spatial language (whether people represent a scene from their own or a different spatial perspective), and perspective taking in action language (the extent to which they simulate an action as though they were performing that action). First, we note that vocabulary is used inconsistently across the spatial and action domains, and propose a more transparent vocabulary that will allow researchers to integrate action- and spatial-perspective taking. Second, we note that embodied theories of language comprehension often make the narrow assumption that understanding action descriptions involves adopting the perspective of an agent carrying out that action. We argue that comprehenders can adopt embodied action-perspectives other than that of the agent, including those of the patient or an observer. Third, we review evidence showing that perspective taking in spatial language is a flexible process. We argue that the flexibility of spatial-perspective taking provides a means for conversation partners engaged in dialogue to maximize similarity between their situation models. These situation models can then be used as the basis for action language simulations, in which language users adopt a particular action-perspective. PMID:24062676

  10. Differential Dynamic and Structural Behavior of Lipid-Cholesterol Domains in Model Membranes

    PubMed Central

    Aguilar, Luis F.; Pino, José A.; Soto-Arriaza, Marco A.; Cuevas, Francisco J.; Sánchez, Susana; Sotomayor, Carlos P.

    2012-01-01

    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). PMID:22768264

  11. Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing*

    PubMed Central

    Pieren, Michel; Desfougères, Yann; Michaillat, Lydie; Schmidt, Andrea; Mayer, Andreas

    2015-01-01

    Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion. PMID:25817997

  12. Plasma membrane domains participate in pH banding of Chara internodal cells.

    PubMed

    Schmölzer, Patric M; Höftberger, Margit; Foissner, Ilse

    2011-08-01

    We investigated the identity and distribution of cortical domains, stained by the endocytic marker FM 1-43, in branchlet internodal cells of the characean green algae Chara corallina and Chara braunii. Co-labeling with NBD C(6)-sphingomyelin, a plasma membrane dye, which is not internalized, confirmed their location in the plasma membrane, and co-labelling with the fluorescent pH indicator Lysotracker red indicated an acidic environment. The plasma membrane domains co-localized with the distribution of an antibody against a proton-translocating ATPase, and electron microscopic data confirmed their identity with elaborate plasma membrane invaginations known as charasomes. The average size and the distribution pattern of charasomes correlated with the pH banding pattern of the cell. Charasomes were larger and more frequent at the acidic regions than at the alkaline bands, indicating that they are involved in outward-directed proton transport. Inhibition of photosynthesis by DCMU prevented charasome formation, and incubation in pH buffers resulted in smaller, homogenously distributed charasomes irrespective of whether the pH was clamped at 5.5 or 8.5. These data indicate that the differential size and distribution of charasomes is not due to differences in external pH but reflects active, photosynthesis-dependent pH banding. The fact that pH banding recovered within several minutes in unbuffered medium, however, confirms that pH banding is also possible in cells with evenly distributed charasomes or without charasomes. Cortical mitochondria were also larger and more abundant at the acid bands, and their intimate association with charasomes and chloroplasts suggests an involvement in carbon uptake and photorespiration. PMID:21659328

  13. Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties

    PubMed Central

    2016-01-01

    Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure–function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer

  14. Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties.

    PubMed

    Heberle, Frederick A; Marquardt, Drew; Doktorova, Milka; Geier, Barbara; Standaert, Robert F; Heftberger, Peter; Kollmitzer, Benjamin; Nickels, Jonathan D; Dick, Robert A; Feigenson, Gerald W; Katsaras, John; London, Erwin; Pabst, Georg

    2016-05-24

    Cell membranes possess a complex three-dimensional architecture, including nonrandom lipid lateral organization within the plane of a bilayer leaflet, and compositional asymmetry between the two leaflets. As a result, delineating the membrane structure-function relationship has been a highly challenging task. Even in simplified model systems, the interactions between bilayer leaflets are poorly understood, due in part to the difficulty of preparing asymmetric model membranes that are free from the effects of residual organic solvent or osmotic stress. To address these problems, we have modified a technique for preparing asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange in order to produce tensionless, solvent-free aLUVs suitable for a range of biophysical studies. Leaflet composition and structure were characterized using isotopic labeling strategies, which allowed us to avoid the use of bulky labels. NMR and gas chromatography provided precise quantification of the extent of lipid exchange and bilayer asymmetry, while small-angle neutron scattering (SANS) was used to resolve bilayer structural features with subnanometer resolution. Isotopically asymmetric POPC vesicles were found to have the same bilayer thickness and area per lipid as symmetric POPC vesicles, demonstrating that the modified exchange protocol preserves native bilayer structure. Partial exchange of DPPC into the outer leaflet of POPC vesicles produced chemically asymmetric vesicles with a gel/fluid phase-separated outer leaflet and a uniform, POPC-rich inner leaflet. SANS was able to separately resolve the thicknesses and areas per lipid of coexisting domains, revealing reduced lipid packing density of the outer leaflet DPPC-rich phase compared to typical gel phases. Our finding that a disordered inner leaflet can partially fluidize ordered outer leaflet domains indicates some degree of interleaflet coupling, and invites speculation on a role for bilayer

  15. Phosphatidylserine-containing membranes alter the thermal stability of prothrombin's catalytic domain: a differential scanning calorimetric study.

    PubMed

    Lentz, B R; Zhou, C M; Wu, J R

    1994-05-10

    Denaturation profiles of bovine prothrombin and its isolated fragments were examined in the presence of Na2EDTA, 5 mM CaCl2, and CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with bovine brain phosphatidylserine (PS). We have shown previously [Lentz, B. R., Wu, J. R., Sorrentino, A. M., & Carleton, J. A. (1991) Biophys. J. 60, 70] that binding to PS/POPC (25/75) large unilamellar vesicles resulted in an enthalpy loss in the main endotherm of prothrombin denaturation (Tm approximately 57-58 degrees C) and a comparable enthalpy gain in a minor endotherm (Tm approximately 59 degrees C) accompanying an upward shift in peak temperature (Tm approximately 73 degrees C). This minor endotherm was also responsive to Ca2+ binding and, in the absence of PS/POPC membranes, corresponded to melting of the N-terminal, Ca2+ and membrane binding domain (fragment 1). Peak deconvolution analysis of the prothrombin denaturation profile and extensive studies of the denaturation of isolated prothrombin domains in the presence and absence of PS/POPC vesicles suggested that membrane binding induced changes in the C-terminal catalytic domain of prothrombin (prethrombin 2) and in a domain that links fragment 1 with the catalytic domain (fragment 2). Specifically, the results have confirmed that the fragment 2 domain interacts with the stabilizes the prethrombin 2 domain and also have shown that fragment 2 interacts directly with the membrane. In addition, the results have demonstrated a heretofore unrecognized interaction between the catalytic and membrane binding domains. This interaction can account for another portion of the denaturation enthalpy that appears at high temperatures in the presence of membranes.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8180168

  16. Porcine reproductive and respiratory syndrome virus nonstructural protein 2 (nsp2) topology and selective isoform integration in artificial membranes.

    PubMed

    Kappes, Matthew A; Miller, Cathy L; Faaberg, Kay S

    2015-07-01

    The membrane insertion and topology of nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) strain VR-2332 was assessed using a cell free translation system in the presence or absence of artificial membranes. Expression of PRRSV nsp2 in the absence of all other viral factors resulted in the genesis of both full-length nsp2 as well as a select number of C-terminal nsp2 isoforms. Addition of membranes to the translation stabilized the translation reaction, resulting in predominantly full-length nsp2 as assessed by immunoprecipitation. Analysis further showed full-length nsp2 strongly associates with membranes, along with two additional large nsp2 isoforms. Membrane integration of full-length nsp2 was confirmed through high-speed density fractionation, protection from protease digestion, and immunoprecipitation. The results demonstrated that nsp2 integrated into the membranes with an unexpected topology, where the amino (N)-terminal (cytoplasmic) and C-terminal (luminal) domains were orientated on opposite sides of the membrane surface. PMID:25768891

  17. Detectors for evaluating the cellular landscape of sphingomyelin- and cholesterol-rich membrane domains.

    PubMed

    Kishimoto, Takuma; Ishitsuka, Reiko; Kobayashi, Toshihide

    2016-08-01

    Although sphingomyelin and cholesterol are major lipids of mammalian cells, the detailed distribution of these lipids in cellular membranes remains still obscure. However, the recent development of protein probes that specifically bind sphingomyelin and/or cholesterol provides new information about the landscape of the lipid domains that are enriched with sphingomyelin or cholesterol or both. Here, we critically summarize the tools to study distribution and dynamics of sphingomyelin and cholesterol. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. PMID:26993577

  18. Eisosomes Are Dynamic Plasma Membrane Domains Showing Pil1-Lsp1 Heteroligomer Binding Equilibrium

    PubMed Central

    Olivera-Couto, Agustina; Salzman, Valentina; Mailhos, Milagros; Digman, Michelle A.; Gratton, Enrico; Aguilar, Pablo S.

    2015-01-01

    Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits. PMID:25863055

  19. Eisosomes are dynamic plasma membrane domains showing pil1-lsp1 heteroligomer binding equilibrium.

    PubMed

    Olivera-Couto, Agustina; Salzman, Valentina; Mailhos, Milagros; Digman, Michelle A; Gratton, Enrico; Aguilar, Pablo S

    2015-04-01

    Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where Pil1-Lsp1 heterodimers are the minimal eisosomes building blocks. Moreover, individual-eisosome fluorescence fluctuation analysis shows that eisosomes in the same cell are not equal domains: while roughly half of them are mostly static, the other half is actively exchanging core protein subunits. PMID:25863055

  20. Expression and purification of integral membrane metallopeptidase HtpX.

    PubMed

    Arolas, Joan L; García-Castellanos, Raquel; Goulas, Theodoros; Akiyama, Yoshinori; Gomis-Rüth, F Xavier

    2014-07-01

    Little is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His8-tag, extracted from the membranes using octyl-β-d-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members. PMID:24769134

  1. An equivalent domain integral method in the two-dimensional analysis of mixed mode crack problems

    NASA Technical Reports Server (NTRS)

    Raju, I. S.; Shivakumar, K. N.

    1990-01-01

    An equivalent domain integral (EDI) method for calculating J-integrals for two-dimensional cracked elastic bodies is presented. The details of the method and its implementation are presented for isoparametric elements. The EDI method gave accurate values of the J-integrals for two mode I and two mixed mode problems. Numerical studies showed that domains consisting of one layer of elements are sufficient to obtain accurate J-integral values. Two procedures for separating the individual modes from the domain integrals are presented.

  2. Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain

    PubMed Central

    Barros, Marilia; Jin, Danni; Lösche, Mathias; Vogt, Volker M.

    2015-01-01

    ABSTRACT The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assembly in vitro dramatically reduced binding of Gag to liposomes. In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize. IMPORTANCE The retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with

  3. Comparative genomic analysis of integral membrane transport proteins in ciliates.

    PubMed

    Kumar, Ujjwal; Saier, Milton H

    2015-01-01

    Integral membrane transport proteins homologous to those found in the Transporter Classification Database (TCDB; www.tcdb.org) were identified and bioinformatically characterized by transporter class, family, and substrate specificity in three ciliates, Paramecium tetraurelia (Para), Tetrahymena thermophila (Tetra), and Ichthyophthirius multifiliis (Ich). In these three organisms, 1,326 of 39,600 proteins (3.4%), 1,017 of 24,800 proteins (4.2%), and 504 out of 8,100 proteins (6.2%) integral membrane transport proteins were identified, respectively. Thus, an inverse relationship was observed between the % transporters identified and the number of total proteins per genome reported. This surprising observation provides insight into the evolutionary process, giving rise to genome reduction following whole genome duplication (as in the case of Para) or during pathogenic association with a host organism (Ich). Of these transport proteins in Para and Tetra, about 41% were channels (more than any other type of organism studied), 31% were secondary carriers (fewer than most eukaryotes) and 26% were primary active transporters, mostly ATP-hydrolysis driven (more than most other eukaryotes). In Ich, the number of channels was selectively reduced by 66%, relative to Para and Tetra. Para has four times more inorganic anion transporters than Tetra, and Ich has nonselectively lost most of these. Tetra and Ich preferentially transport sugars and monocarboxylates while Para prefers di- and tricarboxylates. These observations serve to characterize the transport proteins of these related ciliates, providing insight into their nutrition and metabolism. PMID:25099884

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

    PubMed Central

    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

  5. Evidence for multiple mechanisms for membrane binding and integration via carboxyl-terminal insertion sequences.

    PubMed

    Kim, P K; Janiak-Spens, F; Trimble, W S; Leber, B; Andrews, D W

    1997-07-22

    Subcellular localization of proteins with carboxyl-terminal insertion sequences requires the molecule be both targeted to and integrated into the correct membrane. The mechanism of membrane integration of cytochrome b5 has been shown to be promiscuous, spontaneous, nonsaturable, and independent of membrane proteins. Thus endoplasmic reticulum localization for cytochrome b5 depends primarily on accurate targeting to the appropriate membrane. Here direct comparison of this mechanism with that of three other proteins integrated into membranes via carboxyl-terminal insertion sequences [vesicle-associated membrane protein 1(Vamp1), polyomavirus middle-T antigen, and Bcl-2] revealed that, unlike cytochrome b5, membrane selectivity for these molecules is conferred at least in part by the mechanisms of membrane integration. Bcl-2 membrane integration was similar to that of cytochrome b5 except that insertion into lipid vesicles was inefficient. Unlike cytochrome b5 and Bcl-2, Vamp1 binding to canine pancreatic microsomes was saturable, ATP-dependent, and abolished by mild trypsin treatment of microsomes. Surprisingly, although the insertion sequence of polyomavirus middle-T antigen was sufficient to mediate electrostatic binding to membranes, binding did not lead to integration into the bilayer. Together these results demonstrate that there are at least two different mechanisms for correct membrane integration of proteins with insertion sequences, one mediated primarily by targeting and one relying on factors in the target membrane to mediate selective integration. Our results also demonstrate that, contrary to expectation, hydrophobicity is not sufficient for insertion sequence-mediated membrane integration. We suggest that the structure of the insertion sequence determines whether or not specific membrane-bound receptor proteins are required for membrane integration. PMID:9220974

  6. Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation

    SciTech Connect

    Reider, Amanda; Barker, Sarah L.; Mishra, Sanjay K.; Im, Young Jun; Maldonado-Báez, Lymarie; Hurley, James H.; Traub, Linton M.; Wendland, Beverly

    2010-10-28

    Internalization of diverse transmembrane cargos from the plasma membrane requires a similarly diverse array of specialized adaptors, yet only a few adaptors have been characterized. We report the identification of the muniscin family of endocytic adaptors that is conserved from yeast to human beings. Solving the structures of yeast muniscin domains confirmed the unique combination of an N-terminal domain homologous to the crescent-shaped membrane-tubulating EFC/F-BAR domains and a C-terminal domain homologous to cargo-binding {mu} homology domains ({mu}HDs). In vitro and in vivo assays confirmed membrane-tubulation activity for muniscin EFC/F-BAR domains. The {mu}HD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. The transmembrane protein Mid2, earlier implicated in polarized Rho1 signalling, was identified as a cargo of the yeast adaptor protein. These and other data suggest a model in which the muniscins provide a combined adaptor/membrane-tubulation activity that is important for regulating endocytosis.

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

    PubMed Central

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

    2012-01-01

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

  8. The Membrane-anchoring Domain of Epidermal Growth Factor Receptor Ligands Dictates Their Ability to Operate in Juxtacrine Mode

    SciTech Connect

    Dong, Jianying; Opresko, Lee; Chrisler, William B.; Orr, Galya; Quesenberry, Ryan D.; Lauffenburger, Douglas A.; Wiley, H S.

    2005-06-01

    All ligands of the epidermal growth factor receptor (EGFR) are synthesized as membrane-anchored precursors. Previous work has suggested that some ligands, such as EGF, must be proteolytically released to be active, whereas others, such as heparin binding EGF-like growth factor (HB-EGF) can function while still anchored to the membrane (i.e., juxtacrine signaling). To explore the structural basis for these differences in ligand activity, we engineered a series of membrane-anchored ligands in which the core, receptor-binding domain of EGF was combined with different domains of both EGF and HB-EGF. We found that ligands having the N-terminal extension of EGF could not bind to the EGFR, even when released from the membrane. Ligands lacking an N-terminal extension, but possessing the membrane-anchoring domain of EGF still required proteolytic release for activity, whereas ligands with the membrane anchoring domain of HB-EGF could elicit full biological activity while still membrane anchored. Ligands containing the HB-EGF membrane anchor, but lacking an N-terminal extension, activated EGFR during their transit through the Golgi apparatus . However, cell-mixing experiments and fluorescence resonance energy transfer (FRET) studies showed that juxtacrine signaling typically occurred in trans at the cell surface, at points of cell-cell contact. Our data suggest that the membrane-anchoring domain of ligands selectively controls their ability to participate in juxtacrine signaling and thus, only a subclass of EGFR ligands can act in a juxtacrine mode.

  9. Efficient ethanol recovery from yeast fermentation broth with integrated distillation-membrane process

    EPA Science Inventory

    A hybrid process integrating vapor stripping with vapor compression and vapor permeation membrane separation, termed Membrane Assisted Vapor Stripping (MAVS), was evaluated for recovery and dehydration of ethanol from aqueous solution as an alternative to conventional distillatio...

  10. Fusion Activity of HIV gp41 Fusion Domain is Related to its Secondary Structure and Depth of Membrane Insertion in a Cholesterol-Dependent Fashion

    PubMed Central

    Lai, Alex L.; Moorthy, Anna Eswara; Li, Yinling; Tamm, Lukas K.

    2013-01-01

    The HIV gp41 fusion domain plays a critical role in membrane fusion during viral entry. A thorough understanding of the relationship between the structure and activity of the fusion domain in different lipid environments helps to formulate mechanistic models on how it might function in mediating membrane fusion. The secondary structure of the fusion domain in small liposomes composed of different lipid mixtures was investigated by circular dichroism spectroscopy. In membranes containing less than 30 mol% cholesterol the fusion domain formed an α-helix and in membranes containing equal to or more than 30 mol% cholesterol the fusion domain formed β-sheet secondary structure. EPR spectra of spin-labeled fusion domains also indicated different conformations in membranes with and without cholesterol. Power saturation EPR data were further used to determine the orientation and depth of α-helical fusion domains in lipid bilayers. Fusion and membrane perturbation activities of the gp41 fusion domain were measured by lipid mixing and contents leakage. The fusion domain fused membranes in both its helical and β-sheet forms. High cholesterol, which induced β-sheet, promoted fusion, but acidic lipids, which promoted relatively deep membrane insertion as an α-helix, also induced fusion. The results indicate that the structure of the HIV gp41 fusion domain is plastic and depends critically on the lipid environment. Provided their membrane insertion is deep, α-helical and β-sheet conformations contribute to membrane fusion. PMID:22343048

  11. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments

    PubMed Central

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C.; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-01-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H+-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation. PMID:26272553

  12. Convoluted Plasma Membrane Domains in the Green Alga Chara are Depleted of Microtubules and Actin Filaments.

    PubMed

    Sommer, Aniela; Hoeftberger, Margit; Hoepflinger, Marion C; Schmalbrock, Sarah; Bulychev, Alexander; Foissner, Ilse

    2015-10-01

    Charasomes are convoluted plasma membrane domains in the green alga Chara australis. They harbor H(+)-ATPases involved in acidification of the medium, which facilitates carbon uptake required for photosynthesis. In this study we investigated the distribution of cortical microtubules and cortical actin filaments in relation to the distribution of charasomes. We found that microtubules and actin filaments were largely lacking beneath the charasomes, suggesting the absence of nucleating and/or anchoring complexes or an inhibitory effect on polymerization. We also investigated the influence of cytoskeleton inhibitors on the light-dependent growth and the darkness-induced degradation of charasomes. Inhibition of cytoplasmic streaming by cytochalasin D significantly inhibited charasome growth and delayed charasome degradation, whereas depolymerization of microtubules by oryzalin or stabilization of microtubules by paclitaxel had no effect. Our data indicate that the membrane at the cytoplasmic surface of charasomes has different properties in comparison with the smooth plasma membrane. We show further that the actin cytoskeleton is necessary for charasome growth and facilitates charasome degradation presumably via trafficking of secretory and endocytic vesicles, respectively. However, microtubules are required neither for charasome growth nor for charasome degradation. PMID:26272553

  13. Bipartite Topology of Treponema pallidum Repeat Proteins C/D and I: OUTER MEMBRANE INSERTION, TRIMERIZATION, AND PORIN FUNCTION REQUIRE A C-TERMINAL β-BARREL DOMAIN.

    PubMed

    Anand, Arvind; LeDoyt, Morgan; Karanian, Carson; Luthra, Amit; Koszelak-Rosenblum, Mary; Malkowski, Michael G; Puthenveetil, Robbins; Vinogradova, Olga; Radolf, Justin D

    2015-05-01

    We previously identified Treponema pallidum repeat proteins TprC/D, TprF, and TprI as candidate outer membrane proteins (OMPs) and subsequently demonstrated that TprC is not only a rare OMP but also forms trimers and has porin activity. We also reported that TprC contains N- and C-terminal domains (TprC(N) and TprC(C)) orthologous to regions in the major outer sheath protein (MOSP(N) and MOSP(C)) of Treponema denticola and that TprC(C) is solely responsible for β-barrel formation, trimerization, and porin function by the full-length protein. Herein, we show that TprI also possesses bipartite architecture, trimeric structure, and porin function and that the MOSP(C)-like domains of native TprC and TprI are surface-exposed in T. pallidum, whereas their MOSP(N)-like domains are tethered within the periplasm. TprF, which does not contain a MOSP(C)-like domain, lacks amphiphilicity and porin activity, adopts an extended inflexible structure, and, in T. pallidum, is tightly bound to the protoplasmic cylinder. By thermal denaturation, the MOSP(N) and MOSP(C)-like domains of TprC and TprI are highly thermostable, endowing the full-length proteins with impressive conformational stability. When expressed in Escherichia coli with PelB signal sequences, TprC and TprI localize to the outer membrane, adopting bipartite topologies, whereas TprF is periplasmic. We propose that the MOSP(N)-like domains enhance the structural integrity of the cell envelope by anchoring the β-barrels within the periplasm. In addition to being bona fide T. pallidum rare outer membrane proteins, TprC/D and TprI represent a new class of dual function, bipartite bacterial OMP. PMID:25805501

  14. The Dimer Interface of the Membrane Type 1 Matrix Metalloproteinase Hemopexin Domain

    PubMed Central

    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

    2011-01-01

    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. PMID:21193411

  15. Bending Rigidities and Interdomain Forces in Membranes with Coexisting Lipid Domains.

    PubMed

    Kollmitzer, Benjamin; Heftberger, Peter; Podgornik, Rudolf; Nagle, John F; Pabst, Georg

    2015-06-16

    To precisely quantify the fundamental interactions between heterogeneous lipid membranes with coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains, we performed detailed osmotic stress small-angle x-ray scattering experiments by exploiting the domain alignment in raft-mimicking lipid multibilayers. Performing a Monte Carlo-based analysis allowed us to determine with high reliability the magnitude and functional dependence of interdomain forces concurrently with the bending elasticity moduli. In contrast to previous methodologies, this approach enabled us to consider the entropic undulation repulsions on a fundamental level, without having to take recourse to crudely justified mean-field-like additivity assumptions. Our detailed Hamaker-coefficient calculations indicated only small differences in the van der Waals attractions of coexisting Lo and Ld phases. In contrast, the repulsive hydration and undulation interactions differed significantly, with the latter dominating the overall repulsions in the Ld phase. Thus, alignment of like domains in multibilayers appears to originate from both, hydration and undulation repulsions. PMID:26083923

  16. Bending Rigidities and Interdomain Forces in Membranes with Coexisting Lipid Domains

    PubMed Central

    Kollmitzer, Benjamin; Heftberger, Peter; Podgornik, Rudolf; Nagle, John F.; Pabst, Georg

    2015-01-01

    To precisely quantify the fundamental interactions between heterogeneous lipid membranes with coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains, we performed detailed osmotic stress small-angle x-ray scattering experiments by exploiting the domain alignment in raft-mimicking lipid multibilayers. Performing a Monte Carlo-based analysis allowed us to determine with high reliability the magnitude and functional dependence of interdomain forces concurrently with the bending elasticity moduli. In contrast to previous methodologies, this approach enabled us to consider the entropic undulation repulsions on a fundamental level, without having to take recourse to crudely justified mean-field-like additivity assumptions. Our detailed Hamaker-coefficient calculations indicated only small differences in the van der Waals attractions of coexisting Lo and Ld phases. In contrast, the repulsive hydration and undulation interactions differed significantly, with the latter dominating the overall repulsions in the Ld phase. Thus, alignment of like domains in multibilayers appears to originate from both, hydration and undulation repulsions. PMID:26083923

  17. Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain

    PubMed Central

    Schubert, David A.; Gordo, Susana; Chu, H. Hamlet

    2012-01-01

    The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide–MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation. PMID:23166358

  18. Phosphorylation of the Bin, Amphiphysin, and RSV161/167 (BAR) domain of ACAP4 regulates membrane tubulation.

    PubMed

    Zhao, Xuannv; Wang, Dongmei; Liu, Xing; Liu, Lifang; Song, Zhenwei; Zhu, Tongge; Adams, Gregory; Gao, Xinjiao; Tian, Ruijun; Huang, Yuejia; Chen, Runhua; Wang, Fengsong; Liu, Dong; Yu, Xue; Chen, Yong; Chen, Zhengjun; Teng, Maikun; Ding, Xia; Yao, Xuebiao

    2013-07-01

    ArfGAP With Coiled-Coil, Ankyrin Repeat And PH Domains 4 (ACAP4) is an ADP-ribosylation factor 6 (ARF6) GTPase-activating protein essential for EGF-elicited cell migration. However, how ACAP4 regulates membrane dynamics and curvature in response to EGF stimulation is unknown. Here, we show that phosphorylation of the N-terminal region of ACAP4, named the Bin, Amphiphysin, and RSV161/167 (BAR) domain, at Tyr34 is necessary for EGF-elicited membrane remodeling. Domain structure analysis demonstrates that the BAR domain regulates membrane curvature. EGF stimulation of cells causes phosphorylation of ACAP4 at Tyr34, which subsequently promotes ACAP4 homodimer curvature. The phospho-mimicking mutant of ACAP4 demonstrates lipid-binding activity and tubulation in vitro, and ARF6 enrichment at the membrane is associated with ruffles of EGF-stimulated cells. Expression of the phospho-mimicking ACAP4 mutant promotes ARF6-dependent cell migration. Thus, the results present a previously undefined mechanism by which EGF-elicited phosphorylation of the BAR domain controls ACAP4 molecular plasticity and plasma membrane dynamics during cell migration. PMID:23776207

  19. Membrane-Bound Dynamic Structure of an Arginine-Rich Cell-Penetrating Peptide, the Protein Transduction Domain of HIV TAT, from Solid-State NMR

    PubMed Central

    Su, Yongchao; Waring, Alan J.; Ruchala, Piotr; Hong, Mei

    2010-01-01

    The protein transduction domain of HIV-1 TAT, TAT(48-60), is an efficient cell-penetrating peptide (CPP) that diffuses across the lipid membranes of cells despite eight cationic Arg and Lys residues. To understand its mechanism of membrane translocation against the free energy barrier, we have conducted solid-state NMR experiments to determine the site-specific conformation, dynamics, and lipid interaction of the TAT peptide in anionic lipid bilayers. We found that TAT(48-60) is a highly dynamic and nearly random-coil peptide in the lipid bilayer, and inserts into the membrane-water interface near the glycerol backbone region. Arg-phosphate salt bridge interaction was revealed by short guanidinium-phosphate distances and restricted dynamics of the guanidinium. Together with the observation of strong peptide-water cross peaks in 1H spin diffusion spectra, these results indicate that TAT binding to the membrane-water interface is stabilized not only by electrostatic attraction to the anionic lipids, but also by intermolecular hydrogen bonding with the lipid phosphates and water, which may take the role of intramolecular hydrogen bonds in canonical secondary structures. The random-coil structure of TAT and another CPP, penetratin, suggests that the lack of amphipathic structure is essential for rapid translocation of these Arg-rich CPPs across the lipid membrane without causing permanent damages to the membrane integrity. PMID:20550193

  20. Spacesuit Water Membrane Evaporator Integration with the ISS Extravehicular Mobility

    NASA Technical Reports Server (NTRS)

    Margiott, Victoria; Boyle, Robert

    2014-01-01

    NASA has developed a Solid Water Membrane Evaporation (SWME) to provide cooling for the next generation spacesuit. One approach to increasing the TRL of the system is to incorporate this hardware with the existing EMU. Several integration issues were addressed to support a potential demonstration of the SWME with the existing EMU. Systems analysis was performed to assess the capability of the SWME to maintain crewmember cooling and comfort as a replacement for sublimation. The materials of the SWME were reviewed to address compatibility with the EMU. Conceptual system placement and integration with the EMU via an EVA umbilical system to ensure crew mobility and Airlock egress were performed. A concept of operation for EVA use was identified that is compatible with the existing system. This concept is extensible as a means to provide cooling for the existing EMU. The cooling system of one of the EMUs on orbit has degraded, with the root cause undetermined. Should there be a common cause resident on ISS, this integration could provide a means to recover cooling capability for EMUs on orbit.

  1. 3D Vectorial Time Domain Computational Integrated Photonics

    SciTech Connect

    Kallman, J S; Bond, T C; Koning, J M; Stowell, M L

    2007-02-16

    The design of integrated photonic structures poses considerable challenges. 3D-Time-Domain design tools are fundamental in enabling technologies such as all-optical logic, photonic bandgap sensors, THz imaging, and fast radiation diagnostics. Such technologies are essential to LLNL and WFO sponsors for a broad range of applications: encryption for communications and surveillance sensors (NSA, NAI and IDIV/PAT); high density optical interconnects for high-performance computing (ASCI); high-bandwidth instrumentation for NIF diagnostics; micro-sensor development for weapon miniaturization within the Stockpile Stewardship and DNT programs; and applications within HSO for CBNP detection devices. While there exist a number of photonics simulation tools on the market, they primarily model devices of interest to the communications industry. We saw the need to extend our previous software to match the Laboratory's unique emerging needs. These include modeling novel material effects (such as those of radiation induced carrier concentrations on refractive index) and device configurations (RadTracker bulk optics with radiation induced details, Optical Logic edge emitting lasers with lateral optical inputs). In addition we foresaw significant advantages to expanding our own internal simulation codes: parallel supercomputing could be incorporated from the start, and the simulation source code would be accessible for modification and extension. This work addressed Engineering's Simulation Technology Focus Area, specifically photonics. Problems addressed from the Engineering roadmap of the time included modeling the Auston switch (an important THz source/receiver), modeling Vertical Cavity Surface Emitting Lasers (VCSELs, which had been envisioned as part of fast radiation sensors), and multi-scale modeling of optical systems (for a variety of applications). We proposed to develop novel techniques to numerically solve the 3D multi-scale propagation problem for both the microchip

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

    PubMed Central

    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

  3. Identification of two integral membrane proteins of Plasmodium falciparum

    SciTech Connect

    Smythe, J.A.; Coppel, R.L.; Brown, G.V.; Ramasamy, R.; Kemp, D.J.; Anders, R.F. )

    1988-07-01

    The authors describe the isolation and cloning of two integral membrane protein antigens of Plasmodium falciparum. The antigens were isolated by Triton X-114 temperature-dependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage {lambda}gt11-Amp3 cDNA expression library. Clone Ag512 corresponds to a M{sub r} 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a M{sub r} 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with ({sup 3}H)glucosamine and ({sup 3}H)myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the M{sub r} 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor.

  4. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.

    PubMed

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas Jd; Young, Stephen G; Ploug, Michael

    2016-01-01

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. PMID:26725083

  5. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain

    PubMed Central

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas JD; Young, Stephen G; Ploug, Michael

    2016-01-01

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. DOI: http://dx.doi.org/10.7554/eLife.12095.001 PMID:26725083

  6. Some relationships between membrane phospholipid domains, conformational order, and cell shape in intact human erythrocytes.

    PubMed

    Moore, D J; Gioioso, S; Sills, R H; Mendelsohn, R

    1999-01-01

    A novel method developed in this laboratory [D.J. Moore et al., Biochemistry 35 (1996) 229-235; D.J. Moore et al., Biochemistry 36 (1997) 660-664] to study the conformational order and the propensity for domain formation of specific phospholipids in intact human erythrocytes is extended to two additional species. Acyl chain perdeuterated 1,2-dilauroylphosphatidylethanolamine (diC12PE-d46) was incorporated preferentially (in separate experiments) into the inner leaflet of stomatocytic erythrocytes and into the outer leaflet of echinocytic erythrocytes, while acyl chain perdeuterated 1,2-dipentadecanoylphosphatidylcholine (diC15PC-d58) was incorporated into the outer leaflet of echinocytic erythrocytes. The conformational order and phase behavior of the incorporated molecules were monitored through FT-IR studies of the temperature dependence of the CD2 stretching vibrations. For both diC12PE-d46 and diC15PC-d58, the gel-->liquid crystal phase transition persisted when these lipids were located in the outer leaflet of echinocytic cells, a result indicative of the persistence of phospholipid domains. In each case, the transition widths were broadened compared to the pure lipids, suggestive of either small domains or the presence of additional molecular components within the domains. The conformational order of diC12PE-d46 differed markedly depending on its location and the morphology of the cells. When located predominantly in the inner membrane of stomatocytes, the phase transition of this species was abolished and the conformational order compared with pure lipid vesicles at the same temperature was much lower. The current results along with our previous studies provide a sufficient experimental basis to deduce some general principles of phospholipid conformational order and organization in both normal and shape-altered erythrocytes. PMID:9889394

  7. Plant aquaporins: membrane channels with multiple integrated functions.

    PubMed

    Maurel, Christophe; Verdoucq, Lionel; Luu, Doan-Trung; Santoni, Véronique

    2008-01-01

    Aquaporins are channel proteins present in the plasma and intracellular membranes of plant cells, where they facilitate the transport of water and/or small neutral solutes (urea, boric acid, silicic acid) or gases (ammonia, carbon dioxide). Recent progress was made in understanding the molecular bases of aquaporin transport selectivity and gating. The present review examines how a wide range of selectivity profiles and regulation properties allows aquaporins to be integrated in numerous functions, throughout plant development, and during adaptations to variable living conditions. Although they play a central role in water relations of roots, leaves, seeds, and flowers, aquaporins have also been linked to plant mineral nutrition and carbon and nitrogen fixation. PMID:18444909

  8. Thermodynamic properties of the effector domains of MARTX toxins suggest their unfolding for translocation across the host membrane.

    PubMed

    Kudryashova, Elena; Heisler, David; Zywiec, Andrew; Kudryashov, Dmitri S

    2014-06-01

    MARTX (multifunctional autoprocessing repeats-in-toxin) family toxins are produced by Vibrio cholerae, Vibrio vulnificus, Aeromonas hydrophila and other Gram-negative bacteria. Effector domains of MARTX toxins cross the cytoplasmic membrane of a host cell through a putative pore formed by the toxin's glycine-rich repeats. The structure of the pore is unknown and the translocation mechanism of the effector domains is poorly understood. We examined the thermodynamic stability of the effector domains of V. cholerae and A. hydrophila MARTX toxins to elucidate the mechanism of their translocation. We found that all but one domain in each toxin are thermodynamically unstable and several acquire a molten globule state near human physiological temperatures. Fusion of the most stable cysteine protease domain to the adjacent effector domain reduces its thermodynamic stability ∼ 1.4-fold (from D G H 2 O 21.8 to 16.1 kJ mol(-1) ). Precipitation of several individual domains due to thermal denaturation is reduced upon their fusion into multi-domain constructs. We speculate that low thermostability of the MARTX effector domains correlates with that of many other membrane-penetrating toxins and implies their unfolding for cell entry. This study extends the list of thermolabile bacterial toxins, suggesting that this quality is essential and could be susceptible for selective targeting of pathogenic toxins. PMID:24724536

  9. External push and internal pull forces recruit curvature sensing N-BAR domain proteins to the plasma membrane

    PubMed Central

    Galic, Milos; Jeong, Sangmoo; Tsai, Feng-Chiao; Joubert, Lydia-Marie; Wu, Yi I.; Hahn, Klaus M.; Cui, Yi; Meyer, Tobias

    2012-01-01

    Many of the more than 20 mammalian proteins with N-BAR domains1-2 control cell architecture3 and endocytosis4-5 by associating with curved sections of the plasma membrane (PM)6. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the PM or indirectly by PM-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally-induced inward deformation of the PM by cone-shaped nanostructures (Nanocones) and internally-induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved PM. Markedly, live-cell imaging in adherent cells showed selective recruitment of full length N-BAR proteins and isolated N-BAR domains to PM sub-regions above Nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by Nanocones. We further showed that N-BAR domains are periodically recruited to curved PM sites during local lamellipodia retraction in the front of migrating cells. Recruitment required Myosin II-generated force applied to PM connected actin cables. Together, our study shows that N-BAR domains can be directly recruited to the PM by external push or internal pull forces that locally curve the PM. PMID:22750946

  10. Structure of the Membrane-tethering GRASP Domain Reveals a Unique PDZ Ligand Interaction That Mediates Golgi Biogenesis

    SciTech Connect

    Truschel, S.T.; Heroux, A.; Sengupta, D.; Foote, A.; Macbeth, M. R.; Linstedt, A. D.

    2011-06-10

    Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.

  11. Structure of the Membrane-tethering GRASP Domain Reveals a Unique PDZ Ligand Interaction That Mediates Golgi Biogenesis

    SciTech Connect

    S Truschel; D Sengupta; A Foote; A Heroux; M Macbeth; A Linstedt

    2011-12-31

    Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.

  12. Structure of the Membrane-tethering GRASP Domain Reveals a Unique PDZ Ligand Interaction That Mediates Golgi Biogenesis*

    PubMed Central

    Truschel, Steven T.; Sengupta, Debrup; Foote, Adam; Heroux, Annie; Macbeth, Mark R.; Linstedt, Adam D.

    2011-01-01

    Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae. PMID:21515684

  13. Genome-wide association study for sperm membrane integrity in frozen-thawed semen of Holstein-Friesian bulls.

    PubMed

    Kamiński, Stanisław; Hering, Dorota M; Oleński, Kamil; Lecewicz, Marek; Kordan, Władysław

    2016-07-01

    The aim of the study was to screen the entire bull genome to identify SNP markers and propose candidate genes potentially involved in the variation of sperm membrane integrity in Holstein-Friesian bulls. Two hundred eighty eight bulls kept in one AI center were included in the study. Each bull was genotyped for 54.001 Single Nucleotide Polymorpisms (SNP) by the Illumina BovineSNP50 BeadChip. Commercial straws of frozen-thawed semen were used for the evaluation of sperm plasma membrane integrity (SYBR-14/PI staining) and sperm mitochondrial function (JC1/PI staining). An additive model for Linear Regression Analysis was applied to estimate the effect of SNP marker for sperm membrane integrity (by the use of GoldenHelix SVS7 software). Five significant markers (encompassing 2,2 MB region located on chromosome 6) for SYBR-14/PI were found. Among them one marker-rs41570391 passed Bonferroni correction test. Within approximately 3 Mb genomic region including significant markers three candidate genes: SGMS2 (Sphingomyelin Synthase 2), TET2 (Methylcytosine dioxygenase 2) and GSTCD genes (Gluthatione S-transferase C terminal domain) were proposed as potentially involved in sperm membrane integrity in frozen-thawed semen of Holstein-Friesian bulls. PMID:27236378

  14. The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH

    PubMed Central

    Araye, Anne; Goudet, Amélie; Barbier, Julien; Pichard, Sylvain; Baron, Bruno; England, Patrick; Pérez, Javier; Zinn-Justin, Sophie; Chenal, Alexandre; Gillet, Daniel

    2016-01-01

    Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC. PMID:27070312

  15. Nonlinear conductance and heterogeneity of voltage-gated ion channels allow defining electrical surface domains in cell membranes

    NASA Astrophysics Data System (ADS)

    Cervera, Javier; Manzanares, José A.; Mafe, Salvador

    2015-07-01

    The membrane potential of a cell measured by typical electrophysiological methods is only an average magnitude and experimental techniques allowing a more detailed mapping of the cell surface have shown the existence of spatial domains with locally different electric potentials and currents. Electrical potentials in non-neural cells are regulated by the nonlinear conductance of membrane ion channels. Voltage-gated potassium channels participate in cell hyperpolarization/depolarization processes and control the electrical signals over the cell surface, constituting good candidates to study basic biological questions on a more simplified scale than the complex cell membrane. These channels show also a high heterogeneity, making it possible to analyze the effects of diversity in the electrical responses of channels localized on spatial domains. We use a phenomenological approach of voltage gating that reproduces the observed rectification characteristics of inward rectifying potassium channels and relate the threshold voltage heterogeneity of the channels to the establishment of spatial domains with different electrical sensitivities. Although our model is only a limited picture of the whole cell membrane, it shows that domains with different ion channels may permit or suppress steady state bioelectrical signals over the cell surface according to their particular voltage sensitivity. Also, the nonlinear electrical coupling of channels with different threshold potentials can lead to a rich variety of bioelectrical phenomena, including regions of membrane potential bi-stability.

  16. The F-BAR domain of srGAP2 induces membrane protrusions required for neuronal migration and morphogenesis

    PubMed Central

    Guerrier, Sabrice; Coutinho-Budd, Jaeda; Sassa, Takayuki; Gresset, Aurélie; Jordan, Nicole Vincent; Cheng, Ken; Jin, Wei-Lin; Frost, Adam; Polleux, Franck

    2009-01-01

    SUMMARY During brain development, proper neuronal migration and morphogenesis is critical for the establishment of functional neural circuits. Here we report that srGAP2 negatively regulates neuronal migration and induces neurite outgrowth and branching through the ability of its F-BAR domain to induce filopodia-like membrane protrusions resembling those induced by I-BAR domains in vivo and in vitro. Previous work has suggested that in non-neuronal cells, filopodia dynamics decreases the rate of cell migration and the persistence of leading edge protrusions. srGAP2 knockdown reduces leading process branching and increases the rate of neuronal migration in vivo. Overexpression of srGAP2 or its F-BAR domain has the opposite effects, increasing leading process branching and decreasing migration. These results (1) suggest that F-BAR domains are functionally diverse and (2) highlight the functional importance of proteins directly regulating membrane deformation for proper neuronal migration and morphogenesis. PMID:19737524

  17. Dynamic ergosterol- and ceramide-rich domains in the peroxisomal membrane serve as an organizing platform for peroxisome fusion.

    PubMed

    Boukh-Viner, Tatiana; Guo, Tong; Alexandrian, Alex; Cerracchio, André; Gregg, Christopher; Haile, Sandra; Kyskan, Robert; Milijevic, Svetlana; Oren, Daniel; Solomon, Jonathan; Wong, Vivianne; Nicaud, Jean-Marc; Rachubinski, Richard A; English, Ann M; Titorenko, Vladimir I

    2005-02-28

    We describe unusual ergosterol- and ceramide-rich (ECR) domains in the membrane of yeast peroxisomes. Several key features of these detergent-resistant domains, including the nature of their sphingolipid constituent and its unusual distribution across the membrane bilayer, clearly distinguish them from well characterized detergent-insoluble lipid rafts in the plasma membrane. A distinct set of peroxisomal proteins, including two ATPases, Pex1p and Pex6p, as well as phosphoinositide- and GTP-binding proteins, transiently associates with the cytosolic face of ECR domains. All of these proteins are essential for the fusion of the immature peroxisomal vesicles P1 and P2, the earliest intermediates in a multistep pathway leading to the formation of mature, metabolically active peroxisomes. Peroxisome fusion depends on the lateral movement of Pex1p, Pex6p, and phosphatidylinositol-4,5-bisphosphate-binding proteins from ECR domains to a detergent-soluble portion of the membrane, followed by their release to the cytosol. Our data suggest a model for the multistep reorganization of the multicomponent peroxisome fusion machinery that transiently associates with ECR domains. PMID:15738267

  18. Role of acidic residues in helices TH8-TH9 in membrane interactions of the diphtheria toxin T domain.

    PubMed

    Ghatak, Chiranjib; Rodnin, Mykola V; Vargas-Uribe, Mauricio; McCluskey, Andrew J; Flores-Canales, Jose C; Kurnikova, Maria; Ladokhin, Alexey S

    2015-04-01

    The pH-triggered membrane insertion of the diphtheria toxin translocation domain (T domain) results in transferring the catalytic domain into the cytosol, which is relevant to potential biomedical applications as a cargo-delivery system. Protonation of residues is suggested to play a key role in the process, and residues E349, D352 and E362 are of particular interest because of their location within the membrane insertion unit TH8-TH9. We have used various spectroscopic, computational and functional assays to characterize the properties of the T domain carrying the double mutation E349Q/D352N or the single mutation E362Q. Vesicle leakage measurements indicate that both mutants interact with the membrane under less acidic conditions than the wild-type. Thermal unfolding and fluorescence measurements, complemented with molecular dynamics simulations, suggest that the mutant E362Q is more susceptible to acid destabilization because of disruption of native intramolecular contacts. Fluorescence experiments show that removal of the charge in E362Q, and not in E349Q/D352N, is important for insertion of TH8-TH9. Both mutants adopt a final functional state upon further acidification. We conclude that these acidic residues are involved in the pH-dependent action of the T domain, and their replacements can be used for fine tuning the pH range of membrane interactions. PMID:25875295

  19. Implementation of equivalent domain integral method in the two-dimensional analysis of mixed mode problems

    NASA Technical Reports Server (NTRS)

    Raju, I. S.; Shivakumar, K. N.

    1989-01-01

    An equivalent domain integral (EDI) method for calculating J-intergrals for two-dimensional cracked elastic bodies is presented. The details of the method and its implementation are presented for isoparametric elements. The total and product integrals consist of the sum of an area of domain integral and line integrals on the crack faces. The line integrals vanish only when the crack faces are traction free and the loading is either pure mode 1 or pure mode 2 or a combination of both with only the square-root singular term in the stress field. The EDI method gave accurate values of the J-integrals for two mode I and two mixed mode problems. Numerical studies showed that domains consisting of one layer of elements are sufficient to obtain accurate J-integral values. Two procedures for separating the individual modes from the domain integrals are presented. The procedure that uses the symmetric and antisymmetric components of the stress and displacement fields to calculate the individual modes gave accurate values of the integrals for all problems analyzed. The EDI method when applied to a problem of an interface crack in two different materials showed that the mode 1 and mode 2 components are domain dependent while the total integral is not. This behavior is caused by the presence of the oscillatory part of the singularity in bimaterial crack problems. The EDI method, thus, shows behavior similar to the virtual crack closure method for bimaterial problems.

  20. A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs

    PubMed Central

    Vincent, Patrick; Chua, Michael; Nogue, Fabien; Fairbrother, Ashley; Mekeel, Hal; Xu, Yue; Allen, Nina; Bibikova, Tatiana N.; Gilroy, Simon; Bankaitis, Vytas A.

    2005-01-01

    Phosphatidylinositol (PtdIns) transfer proteins (PITPs) regulate signaling interfaces between lipid metabolism and membrane trafficking. Herein, we demonstrate that AtSfh1p, a member of a large and uncharacterized Arabidopsis thaliana Sec14p-nodulin domain family, is a PITP that regulates a specific stage in root hair development. AtSfh1p localizes along the root hair plasma membrane and is enriched in discrete plasma membrane domains and in the root hair tip cytoplasm. This localization pattern recapitulates that visualized for PtdIns(4,5)P2 in developing root hairs. Gene ablation experiments show AtSfh1p nullizygosity compromises polarized root hair expansion in a manner that coincides with loss of tip-directed PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, and manifest disorganization of the root hair microtubule cytoskeleton. Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu. We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex. We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants. PMID:15728190

  1. Integrative curriculum reform, domain dependent knowing, and teachers` epistemological theories: Implications for middle-level teaching

    SciTech Connect

    Powell, R.R.

    1998-12-01

    Integrative curriculum as both a theoretical construct and a practical reality, and as a theme-based, problem-centered, democratic way of schooling, is becoming more widely considered as a feasible alternative to traditional middle-level curricula. Importantly for teaching and learning, domain dependence requires teachers to view one area of knowledge as fully interdependent with other areas of knowledge during the learning process. This requires teachers to adopt personal epistemological theories that reflect integrative, domain dependent knowing. This study explored what happened when teachers from highly traditional domain independent school settings encountered an ambitious college-level curriculum project that was designed to help the teachers understand the potential that integrative, domain dependent teaching holds for precollege settings. This study asked: What influence does an integrative, domain dependent curriculum project have on teachers` domain independent, epistemological theories for teaching and learning? Finding an answer to this question is essential if we, as an educational community, are to understand how integrative curriculum theory is transformed by teachers into systemic curriculum reform. The results suggest that the integrative curriculum project that teachers participated in did not explicitly alter their classroom practices in a wholesale manner. Personal epistemological theories of teachers collectively precluded teachers from making any wholesale changes in their individual classroom teaching. However, teachers became aware of integrative curriculum as an alternative, and they expressed interest in infusing integrative practices into their classrooms as opportunities arise.

  2. On the interactions between nucleotide binding domains and membrane spanning domains in cystic fibrosis transmembrane regulator: A molecular dynamic study.

    PubMed

    Belmonte, Luca; Moran, Oscar

    2015-04-01

    The Cystic Fibrosis Transmembrane Regulator (CFTR) is a membrane protein whose mutations cause cystic fibrosis, a lethal genetic disease. We performed a molecular dynamic (MD) study of the properties of the nucleotide binding domains (NBD) whose conformational changes, upon ATP binding, are the direct responsible of the gating mechanisms of CFTR. This study was done for the wild type (WT) CFTR and for the two most common mutations, ΔF508, that produces a traffic defect of the protein, and the mutation G551D, that causes a gating defect on CFTR. Using an homology model of the open channel conformation of the CFTR we thus introduced the mutations to the structure. Although the overall structures of the G551D and ΔF508 are quite well conserved, the NBD1-NBD2 interactions are severely modified in both mutants. NBD1 and NBD2 are indeed destabilized with a higher internal energy (Ei) in the ΔF508-CFTR. Differently, Ei does not change in the NBDs of G551D but, while the number of close contacts between NBD1 and NBD2 in ΔF508 is increased, a significant reduction of close contacts is found in the G551D mutated form. Hydrogen bonds formation between NBDs of the two mutated forms is also altered and it is slightly increased for the ΔF508, while are severely reduced in G551D. A consequent modification of the NBDs-ICLs interactions between residues involved in the transduction of the ATP binding and the channel gating is also registered. Indeed, while a major interaction is noticed between NBDs interface and ICL2 and ICL4 in the WT, this interaction is somehow altered in both mutated forms plausibly with effect on channel gating. Thus, single point mutations of the CFTR protein can reasonably results in channel gating defects due to alteration of the interaction mechanisms between the NBDs and NBDs-ICLs interfaces upon ATP-binding process. PMID:25640670

  3. Effects of Bloom-Forming Algae on Fouling of Integrated Membrane Systems in Seawater Desalination

    ERIC Educational Resources Information Center

    Ladner, David Allen

    2009-01-01

    Combining low- and high-pressure membranes into an integrated membrane system is an effective treatment strategy for seawater desalination. Low-pressure microfiltration (MF) and ultrafiltration (UF) membranes remove particulate material, colloids, and high-molecular-weight organics leaving a relatively foulant-free salt solution for treatment by…

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

    PubMed Central

    Weber, Kathrin; Harper, Nicholas; Schwabe, John; Cohen, Gerald M.

    2013-01-01

    Summary 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. PMID:24120870

  5. Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells

    PubMed Central

    Rossotti, Martín; Tabares, Sofía; Alfaya, Lucía; Leizagoyen, Carmen; Moron, Gabriel; González-Sapienza, Gualberto

    2015-01-01

    BACKGROUND Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domain (nanobody) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct used for pull-down/MS target identification. RESULTS The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS This strategy streamline the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets. PMID:25819371

  6. Gramicidin Alters the Lipid Compositions of Liquid-Ordered and Liquid-Disordered Membrane Domains

    NASA Astrophysics Data System (ADS)

    Hassan-Zadeh, Ebrahim; Huang, Juyang

    2012-10-01

    The effects of adding 1 mol % of gramicidin A to the well-known DOPC/DSPC/cholesterol lipid mixtures were investigated. 4-component giant unilamellar vesicles (GUV) were prepared using our recently developed Wet-Film method. The phase boundary of liquid-ordered and liquid-disordered (Lo-Ld) coexisting region was determined using video fluorescence microscopy. We found that if cares were not taken, light-induced domain artifacts could significantly distort the measured phase boundary. After testing several fluorescence dyes, we found that the emission spectrum of Nile Red is quite sensitive to membrane composition. By fitting the Nile Red emission spectra at the phase boundary to the spectra in the Lo-Ld coexisting region, the thermodynamic tie-lines were determined. As an active component of lipid membranes, gramicidin not only partitions favorably into the liquid-disordered (Ld) phase, it also alters the phase boundary and thermodynamic tie-lines. Even at as low as 1 mol %, gramicidin decreases the cholesterol mole fraction of Ld phase and increases the area of Lo phase.

  7. Ordered and disordered phospholipid domains coexist in membranes containing the calcium pump protein of sarcoplasmic reticulum.

    PubMed Central

    Lentz, B R; Clubb, K W; Barrow, D A; Meissner, G

    1983-01-01

    Data are presented that lead to an alternative model for the organization and molecular dynamics of lipid molecules near the Ca2+-stimulated, Mg2+-dependent adenosinetriphosphatase (Ca2+-ATPase; ATP phosphohydrolase, EC 3.6.1.3) of sarcoplasmic reticulum. Measurements of the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene in progressively delipidated sarcoplasmic reticulum membranes have been quantitatively interpreted in terms of a layer of lipid of high anisotropy (the lipid annulus) coexisting with lipid layers of very low anisotropy. In addition, the Ca2+-ATPase has been reconstituted into pure 1,2-dipentadecanoyl 3-sn-phosphatidylcholine membranes over a range of lipid-to-protein ratios. High-sensitivity differential scanning calorimetry has demonstrated that roughly 30 lipid molecules per Ca2+-ATPase molecule (annular lipids) fail to undergo a calorimetrically detectable phase transition in the temperature range 4-44 degrees C. Roughly 100 lipid molecules beyond the annulus undergo a detectable phase transition at a temperature below the phase transition of pure lipid and with an enthalpy change [4.2 kcal/mol (1 kcal = 4.18 kJ)] about half that observed for pure lipid vesicles (7.7-7.8 kcal/mol). We propose that both the fluorometric and calorimetric data are consistent with a model in which a motionally inhibited lipid annulus is surrounded by a more extensive region of disrupted lipid packing order, which we have called the secondary lipid domain. PMID:6222375

  8. Membrane-inserted conformation of transmembrane domain 4 of divalent-metal transporter.

    PubMed Central

    Li, Hongyan; Li, Fei; Sun, Hongzhe; Qian, Zhong Ming

    2003-01-01

    Divalent-metal transporter 1 (DMT1) is involved in the intestinal iron absorption and in iron transport in the transferrin cycle. It transports metal ions at low pH ( approximately 5.5), but not at high pH (7.4), and the transport is a proton-coupled process. Previously it has been shown that transmembrane domain 4 (TM4) is crucial for the function of this protein. Here we provide the first direct experimental evidence for secondary-structural features and membrane insertions of a 24-residue peptide, corresponding to TM4 of DMT1 (DMTI-TM4), in various membrane-mimicking environments by the combined use of CD and NMR spectroscopies. The peptide mainly adopts an alpha-helical structure in trifluoroethanol, SDS and dodecylphosphocholine micelles, and dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol small unilamellar vesicles. It has been demonstrated from both Halpha secondary shifts and nuclear-Overhauser-enhancement (NOE) connectivities that the peptide is well folded into an alpha-helix from Val(8) to Lys(23) in SDS micelles at pH 4.0, whereas the N-terminus is highly flexible. The alpha-helical content estimated from NMR data is in agreement with that extracted from CD simulations. The highest helicity was observed in the anionic phospholipids [1,2-dimyristoyl- sn -glycero-3-[phospho-rac -(1-glycerol)

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

    PubMed Central

    Kuriyan, Ajay E.; DeBuc, Delia Cabrera; Smiddy, William E.

    2016-01-01

    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. PMID:26949617

  10. MUNI Ways and Structures Building Integrated Solar Membrane Project

    SciTech Connect

    Smith, Randall

    2014-07-03

    The initial goal of the MUNI Ways and Structures Building Integrated Solar Membrane Installation Project was for the City and County of San Francisco (CCSF) to gain experience using the integrated higher efficiency solar photovoltaic (PV) single-ply membrane product, as it differs from the conventional, low efficiency, thin-film PV products, to determine the feasibility of success of larger deployment. As several of CCSF’s municipal rooftops are constrained with respect to weight restrictions, staff of the Energy Generation Group of the San Francisco Public Utilities Commission (SFPUC) proposed to install a solar PV system using single-ply membrane The installation of the 100 kW (DC-STC) lightweight photo voltaic (PV) system at the MUNI Ways and Structures Center (700 Pennsylvania Ave., San Francisco) is a continuation of the commitment of the City and County of San Francisco (CCSF) to increase the pace of municipal solar development, and serve its municipal facilities with clean renewable energy. The fourteen (14) solar photovoltaic systems that have already been installed at CCSF municipal facilities are assisting in the reduction of fossil-fuel use, and reduction of greenhouse gases from fossil combustion. The MUNI Ways & Structures Center roof has a relatively low weight-bearing capacity (3.25 pounds per square foot) and use of traditional crystalline panels was therefore rejected. Consequently it was decided to use the best available highest efficiency Building-Integrated PV (BIPV) technology, with consideration for reliability and experience of the manufacturer which can meet the low weight-bearing capacity criteria. The original goal of the project was to provide an opportunity to monitor the results of the BIPV technology and compare these results to other City and County of San Francisco installed PV systems. The MUNI Ways and Structures Center was acquired from the Cookson Doors Company, which had run the Center for many decades. The building was

  11. The inverse BAR domain protein IBARa drives membrane remodeling to control osmoregulation, phagocytosis and cytokinesis.

    PubMed

    Linkner, Joern; Witte, Gregor; Zhao, Hongxia; Junemann, Alexander; Nordholz, Benjamin; Runge-Wollmann, Petra; Lappalainen, Pekka; Faix, Jan

    2014-03-15

    Here, we analyzed the single inverse Bin/Amphiphysin/Rvs (I-BAR) family member IBARa from Dictyostelium discoideum. The X-ray structure of the N-terminal I-BAR domain solved at 2.2 Å resolution revealed an all-α-helical structure that self-associates into a 165-Å zeppelin-shaped antiparallel dimer. The structural data are consistent with its shape in solution obtained by small-angle X-ray scattering. Cosedimentation, fluorescence anisotropy, and fluorescence and electron microscopy revealed that the I-BAR domain bound preferentially to phosphoinositide-containing vesicles and drove the formation of negatively curved tubules. Immunofluorescence labeling further showed accumulation of endogenous IBARa at the tips of filopodia, the rim of constricting phagocytic cups, in foci connecting dividing cells during the final stage of cytokinesis and most prominently at the osmoregulatory contractile vacuole (CV). Consistently, IBARa-null mutants displayed defects in CV formation and discharge, growth, phagocytosis and mitotic cell division, whereas filopodia formation was not compromised. Of note, IBARa-null mutants were also strongly impaired in cell spreading. Taken together, these data suggest that IBARa constitutes an important regulator of numerous cellular processes intimately linked with the dynamic rearrangement of cellular membranes. PMID:24463811

  12. The Taz1p transacylase is imported and sorted into the outer mitochondrial membrane via a membrane anchor domain.

    PubMed

    Herndon, Jenny D; Claypool, Steven M; Koehler, Carla M

    2013-12-01

    Mutations in the mitochondrial transacylase tafazzin, Taz1p, in Saccharomyces cerevisiae cause Barth syndrome, a disease of defective cardiolipin remodeling. Taz1p is an interfacial membrane protein that localizes to both the outer and inner membranes, lining the intermembrane space. Pathogenic point mutations in Taz1p that alter import and membrane insertion result in accumulation of monolysocardiolipin. In this study, we used yeast as a model to investigate the biogenesis of Taz1p. We show that to achieve this unique topology in mitochondria, Taz1p follows a novel import pathway in which it crosses the outer membrane via the translocase of the outer membrane and then uses the Tim9p-Tim10p complex of the intermembrane space to insert into the mitochondrial outer membrane. Taz1p is then transported to membranes of an intermediate density to reach a location in the inner membrane. Moreover, a pathogenic mutation within the membrane anchor (V224R) alters Taz1p import so that it bypasses the Tim9p-Tim10p complex and interacts with the translocase of the inner membrane, TIM23, to reach the matrix. Critical targeting information for Taz1p resides in the membrane anchor and flanking sequences, which are often mutated in Barth syndrome patients. These studies suggest that altering the mitochondrial import pathway of Taz1p may be important in understanding the molecular basis of Barth syndrome. PMID:24078306

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

    PubMed Central

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

    1992-01-01

    Two experimental techniques have been utilized to explore the barrier properties of lecithin/decane bilayer membranes with the aim of determining the contributions of various domains within the bilayer to the overall barrier. The thickness of lecithin/decane bilayers was systematically varied by modulating the chemical potential of decane in the annulus surrounding the bilayer using different mole fractions of squalene in decane. The dependence of permeability of a model permeant (acetamide) on the thickness of the solvent-filled region of the bilayer was assessed in these bilayers to determine the contribution of this region to the overall barrier. The flux of acetamide was found to vary linearly with bilayer area with Pm = (2.9 +/- 0.3) x 10(-4) cm s-1, after correcting for diffusion through unstirred water layers. The ratio between the overall membrane permeability coefficient and that calculated for diffusion through the hydrocarbon core in membranes having maximum thickness was 0.24, suggesting that the solvent domain contributes only slightly to the overall barrier properties. Consistent with these results, the permeability of acetamide was found to be independent of bilayer thickness. The relative contributions of the bilayer interface and ordered hydrocarbon regions to the transport barrier may be evaluated qualitatively by exploring the effective chemical nature of the barrier microenvironment. This may be probed by comparing functional group contributions to transport with those obtained for partitioning between water and various model bulk solvents ranging in polarity or hydrogen-bonding potential. A novel approach is described for obtaining group contributions to transport using ionizable permeants and pH adjustment. Using this approach, bilayer permeability coefficients of p-toluic acid and p-hydroxymethyl benzoic acid were determined to be 1.1 +/- 0.2 cm s-1 and (1.6 +/- 0.4) x 10(-3) cm s-1, respectively. From these values, the -OH group contribution

  14. Phase separation in biological membranes: integration of theory and experiment

    PubMed Central

    Elson, Elliot L.; Fried, Eliot; Dolbow, John E.; Genin, Guy M.

    2013-01-01

    Lipid bilayer model membranes can undergo transitions between ordered and disordered phases, and membranes that contain a mixture of lipid species can undergo phase separations. Studies of these transformations are of interest for what they can tell us about the interaction energies of lipid molecules of different species and conformations. Nanoscopic phases can provide a model for membrane rafts, which have important biological functions in cell membranes. Important questions are whether lipid nanodomains can exist in stable equilibrium in membranes and what is the distribution of their sizes in membranes of different composition. It is also important to know the lifetimes of nanodomains. Theoretical methods have supplied much important information on these questions, but better experimental methods are needed to detect and characterize nanodomains under normal membrane conditions. This review summarizes linkages between theoretical and experimental studies of phase separation in lipid bilayer model membranes. PMID:20192775

  15. Process Intensification with Integrated Water-Gas-Shift Membrane Reactor

    SciTech Connect

    2009-11-01

    This factsheet describes a research project whose objective is to develop hydrogen-selective membranes for an innovative gas-separation process based on a water-gas-shift membrane reactor (WGS-MR) for the production of hydrogen.

  16. Multiple integral representation for the trigonometric SOS model with domain wall boundaries

    NASA Astrophysics Data System (ADS)

    Galleas, W.

    2012-05-01

    Using the dynamical Yang-Baxter algebra we derive a functional equation for the partition function of the trigonometric SOS model with domain wall boundary conditions. The solution of the equation is given in terms of a multiple contour integral.

  17. AERIS: An Integrated Domain Information System for Aerospace Science and Technology

    ERIC Educational Resources Information Center

    Hatua, Sudip Ranjan; Madalli, Devika P.

    2011-01-01

    Purpose: The purpose of this paper is to discuss the methodology in building an integrated domain information system with illustrations that provide proof of concept. Design/methodology/approach: The present work studies the usual search engine approach to information and its pitfalls. A methodology was adopted for construction of a domain-based…

  18. Membrane anchoring domain of herpes simplex virus glycoprotein gB is sufficient for nuclear envelope localization.

    PubMed Central

    Gilbert, R; Ghosh, K; Rasile, L; Ghosh, H P

    1994-01-01

    We have used the glycoprotein gB of herpes simplex virus type 1 (gB-1), which buds from the inner nuclear membrane, as a model protein to study localization of membrane proteins in the nuclear envelope. To determine whether specific domains of gB-1 glycoprotein are involved in localization in the nuclear envelope, we have used deletion mutants of gB-1 protein as well as chimeric proteins constructed by replacing the domains of the cell surface glycoprotein G of vesicular stomatitis virus with the corresponding domains of gB. Mutant and chimeric proteins expressed in COS cells were localized by immunoelectron microscopy. A chimeric protein (gB-G) containing the ectodomain of gB and the transmembrane and cytoplasmic domains of G did not localize in the nuclear envelope. When the ectodomain of G was fused to the transmembrane and cytoplasmic domains of gB, however, the resulting chimeric protein (G-gB) was localized in the nuclear envelope. Substitution of the transmembrane domain of G with the 69 hydrophobic amino acids containing the membrane anchoring domain of gB allowed the hybrid protein (G-tmgB) to be localized in the nuclear envelope, suggesting that residues 721 to 795 of gB can promote retention of proteins in the nuclear envelope. Deletion mutations in the hydrophobic region further showed that a transmembrane segment of 21 hydrophobic amino acids, residues 774 to 795 of gB, was sufficient for localization in the nuclear envelope. Since wild-type gB and the mutant and chimeric proteins that were localized in the nuclear envelope were also retained in the endoplasmic reticulum, the membrane spanning segment of gB could also influence retention in the endoplasmic reticulum. Images PMID:8139012

  19. Misfolded membrane proteins are specifically recognized by the transmembrane domain of the Hrd1p ubiquitin ligase

    PubMed Central

    Sato, Brian K.; Schulz, Daniel; Do, Phong H.

    2009-01-01

    Summary Quality control pathways such as ER-associated degradation (ERAD) employ a small number of factors to specifically recognize a wide variety of protein substrates. Delineating the mechanisms of substrate selection is a principle goal in studying quality control. The Hrd1p ubiquitin ligase mediates ERAD of numerous misfolded proteins including soluble, lumenal ERAD-L and membrane-anchored ERAD-M substrates. We tested if the Hrd1p multi-spanning membrane domain was involved in ERAD-M specificity. In this work, we have identified site-directed membrane domain mutants of Hrd1p impaired only for ERAD-M and normal for ERAD-L. Furthermore, other Hrd1p variants were specifically deficient for degradation of individual ERAD-M substrates. Thus, the Hrd1p transmembrane region bears determinants of high specificity in the ERAD-M pathway. From in vitro and interaction studies, we suggest a model in which the Hrd1p membrane domain employs intra-membrane residues to evaluate substrate misfolding, leading to selective ubiquitination of appropriate ERAD-M clients. PMID:19394298

  20. Calcium Causes a Conformational Change in Lamin A Tail Domain that Promotes Farnesyl-Mediated Membrane Association

    PubMed Central

    Kalinowski, Agnieszka; Qin, Zhao; Coffey, Kelli; Kodali, Ravi; Buehler, Markus J.; Lösche, Mathias; Dahl, Kris Noel

    2013-01-01

    Lamin proteins contribute to nuclear structure and function, primarily at the inner nuclear membrane. The posttranslational processing pathway of lamin A includes farnesylation of the C-terminus, likely to increase membrane association, and subsequent proteolytic cleavage of the C-terminus. Hutchinson Gilford progeria syndrome is a premature aging disorder wherein a mutant version of lamin A, Δ50 lamin A, retains its farnesylation. We report here that membrane association of farnesylated Δ50 lamin A tail domains requires calcium. Experimental evidence and molecular dynamics simulations collectively suggest that the farnesyl group is sequestered within a hydrophobic region in the tail domain in the absence of calcium. Calcium binds to the tail domain with an affinity KD ≈ 250 μM where it alters the structure of the Ig-fold and increases the solvent accessibility of the C-terminus. In 2 mM CaCl2, the affinity of the farnesylated protein to a synthetic membrane is KD ≈ 2 μM, as measured with surface plasmon resonance, but showed a combination of aggregation and binding. Membrane binding in the absence of calcium could not be detected. We suggest that a conformational change induced in Δ50 lamin A with divalent cations plays a regulatory role in the posttranslational processing of lamin A, which may be important in disease pathogenesis. PMID:23708364

  1. Helical image reconstruction of the outward-open human erythrocyte band 3 membrane domain in tubular crystals.

    PubMed

    Yamaguchi, Tomohiro; Fujii, Takashi; Abe, Yoshito; Hirai, Teruhisa; Kang, Dongchon; Namba, Keiichi; Hamasaki, Naotaka; Mitsuoka, Kaoru

    2010-03-01

    The C-terminal membrane domain of erythrocyte band 3 functions as an anion exchanger. Here, we report the three-dimensional (3D) structure of the membrane domain in an inhibitor-stabilized, outward-open conformation at 18A resolution. Unstained, frozen-hydrated tubular crystals containing the membrane domain of band 3 purified from human red blood cells (hB3MD) were examined using cryo-electron microscopy and iterative helical real-space reconstruction (IHRSR). The 3D image reconstruction of the tubular crystals showed the molecular packing of hB3MD dimers with dimensions of 60 x 110 A in the membrane plane and a thickness of 70A across the membrane. Immunoelectron microscopy and carboxyl-terminal digestion demonstrated that the intracellular surface of hB3MD was exposed on the outer surface of the tubular crystal. A 3D density map revealed that hB3MD consists of at least two subdomains and that the outward-open form is characterized by a large hollow area on the extracellular surface and continuous density on the intracellular surface. PMID:20005958

  2. Calpain cleavage of the B isoform of Ins(1,4,5)P3 3-kinase separates the catalytic domain from the membrane anchoring domain.

    PubMed Central

    Pattni, Krupa; Millard, Thomas H; Banting, George

    2003-01-01

    Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] is one of the key intracellular second messengers in cells and mobilizes Ca2+ stores in the ER (endoplasmic reticulum). Ins(1,4,5)P3 has a short half-life within the cell, and is rapidly metabolized through one of two pathways, one of which involves further phosphorylation of the inositol ring: Ins(1,4,5)P3 3-kinase (IP3-3K) phosphorylates Ins(1,4,5)P3, resulting in the formation of inositol (1,3,4,5)-tetrakisphosphate [Ins(1,3,4,5)P4]. There are three known isoforms of IP3-3K, designated IP3-3KA, IP3-3KB and IP3-3KC. These have differing N-termini, but highly conserved C-termini harbouring the catalytic domain. The three IP3-3K isoforms have different subcellular locations and the B-kinase is uniquely present in both cytosolic and membrane-bound pools. As it is the N-terminus of the B-kinase that differs most from the A- and C-kinases, we have hypothesized that this portion of the protein may be responsible for membrane localization. Although there are no known membrane-targeting protein motifs within the sequence of IP3-3KB, it is found to be tightly associated with the ER membrane. Here, we show that specific regions of the N-terminus of IP3-3KB are necessary and sufficient for efficient membrane localization of the protein. We also report that, in the presence of Ca2+, the kinase domain of IP3-3KB is cleaved from the membrane-anchoring region by calpain. PMID:12906709

  3. The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

    PubMed Central

    Richardson, Brian C; Halaby, Steve L; Gustafson, Margaret A; Fromme, J Christopher

    2016-01-01

    The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth. DOI: http://dx.doi.org/10.7554/eLife.12411.001 PMID:26765562

  4. Air separation by integrally asymmetric hollow-fiber membranes

    SciTech Connect

    Feng, X.; Ivory, J.; Rajan, V.S.V.

    1999-10-01

    Integrally asymmetric hollow-fiber membranes each with an outer skin layer and a porous substrate were studied for air separation to produce nitrogen and oxygen enriched air. The test on both bore-side feed and shell-side feed with concurrent and countercurrent flow arrangements for a wide range of stage cuts shows that the bore-side feed countercurrent flow was the most advantageous configuration in the permeator design. When operated in the bore-side feed countercurrent configuration, the permeator performance compared favorably with the commercial systems available for nitrogen production. A mathematical model was developed for this configuration. Since the concentration polarization in the substrate was a major concern for the bore-side feed configuration, especially for high stage-cut operations, a theoretical approach was pursued to formulate the concentration polarization. This allows for the diagnosis of the significance of concentration polarization in a specific permeation process, although it is difficult to predict concentration polarization accurately due to limited knowledge of the detailed membrance structure.

  5. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

    PubMed Central

    Chen, Xin; Li, Wenjuan; Ren, Junming; Huang, Deli; He, Wan-ting; Song, Yunlong; Yang, Chao; Li, Wanyun; Zheng, Xinru; Chen, Pengda; Han, Jiahuai

    2014-01-01

    Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture. PMID:24366341

  6. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity.

    PubMed

    Murashko, Oleg N; Kaberdin, Vladimir R; Lin-Chao, Sue

    2012-05-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane-protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E-membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1-499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (K(d)) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the "large" domain (amino acids 1-400, consisting of the RNase H, S1, 5'-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E-membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  7. KINETIC INTERMEDIATE REVEALS STAGGERED PH-DEPENDENT TRANSITIONS ALONG THE MEMBRANE INSERTION PATHWAY OF DIPHTHERIA TOXIN T-DOMAIN

    PubMed Central

    Kyrychenko, Alexander; Posokhov, Yevgen O.; Rodnin, Mykola V.; Ladokhin, Alexey S.

    2009-01-01

    The pH-triggered membrane insertion pathway of the T-domain of diphtheria toxin was studied using site-selective fluorescence labeling with subsequent application of several spectroscopic techniques (e.g., fluorescence correlation spectroscopy, FRET, lifetime quenching and kinetic fluorescence). FCS measurements indicate that pH-dependent formation of the membrane-competent form depends only slightly on the amount of anionic lipids in the membrane. The subsequent transbilayer insertion, however, is strongly favored by anionic lipids. Kinetic FRET measurements between donor-labeled T-domain and acceptor-labeled lipid vesicles demonstrate rapid membrane association at all pH values for which binding occurs. In contrast, the transmembrane insertion kinetics is significantly slower, and is also both pH- and lipid-dependent. Analysis of kinetic behavior of binding and insertion indicates the presence of several interfacial intermediates on the insertion pathway of the T-domain, from soluble W-state to transmembrane T-state. Intermediate interfacial I-state can be trapped in membranes with low content of anionic lipids (10%). In membranes of greater anionic lipid content, another pH-dependent transition results in the formation of the insertion-competent state and subsequent transmembrane insertion. Comparison of the results of various kinetic and equilibrium experiments suggests that the pH-dependences determining membrane association and transbilayer insertion transitions are different, but staggered. Anionic lipids not only assist in formation of the insertion competent form, but also lower the kinetic barrier for the final insertion. PMID:19588969

  8. Membrane curvature in cell biology: An integration of molecular mechanisms.

    PubMed

    Jarsch, Iris K; Daste, Frederic; Gallop, Jennifer L

    2016-08-15

    Curving biological membranes establishes the complex architecture of the cell and mediates membrane traffic to control flux through subcellular compartments. Common molecular mechanisms for bending membranes are evident in different cell biological contexts across eukaryotic phyla. These mechanisms can be intrinsic to the membrane bilayer (either the lipid or protein components) or can be brought about by extrinsic factors, including the cytoskeleton. Here, we review examples of membrane curvature generation in animals, fungi, and plants. We showcase the molecular mechanisms involved and how they collaborate and go on to highlight contexts of curvature that are exciting areas of future research. Lessons from how membranes are bent in yeast and mammals give hints as to the molecular mechanisms we expect to see used by plants and protists. PMID:27528656

  9. Outer domains of integrase within retroviral intasomes are dispensible for catalysis of DNA integration.

    PubMed

    Li, Min; Lin, Shiqiang; Craigie, Robert

    2016-02-01

    Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) comprising a pair of viral DNA ends synapsed by a tetramer of integrase. Current integrase inhibitors act on intasomes rather than free integrase protein. Structural and functional studies of intasomes are essential to understand their mechanism of action and how the virus can escape by mutation. To date, prototype foamy virus (PFV) is the only retrovirus for which high-resolution structures of intasomes have been determined. In the PFV intasome structure, only the core domains of the outer subunits are ordered; the N-terminal domain, C-terminal domain, and N-terminal extension domain are disordered. Are these "missing domains" required for function or are they dispensable? We have devised a strategy to assemble "hetero-intasomes" in which the outer domains are not present as a tool to assess the functional role of the missing domains for catalysis of integration. We find that the disordered domains of outer subunits are not required for intasome assembly or catalytic activity as catalytic core domains can substitute for the outer subunits in the case of both PFV and HIV-1 intasomes. PMID:26537415

  10. An Integrated Framework Advancing Membrane Protein Modeling and Design

    PubMed Central

    Weitzner, Brian D.; Duran, Amanda M.; Tilley, Drew C.; Elazar, Assaf; Gray, Jeffrey J.

    2015-01-01

    Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design. PMID:26325167

  11. An Integrated Framework Advancing Membrane Protein Modeling and Design.

    PubMed

    Alford, Rebecca F; Koehler Leman, Julia; Weitzner, Brian D; Duran, Amanda M; Tilley, Drew C; Elazar, Assaf; Gray, Jeffrey J

    2015-09-01

    Membrane proteins are critical functional molecules in the human body, constituting more than 30% of open reading frames in the human genome. Unfortunately, a myriad of difficulties in overexpression and reconstitution into membrane mimetics severely limit our ability to determine their structures. Computational tools are therefore instrumental to membrane protein structure prediction, consequently increasing our understanding of membrane protein function and their role in disease. Here, we describe a general framework facilitating membrane protein modeling and design that combines the scientific principles for membrane protein modeling with the flexible software architecture of Rosetta3. This new framework, called RosettaMP, provides a general membrane representation that interfaces with scoring, conformational sampling, and mutation routines that can be easily combined to create new protocols. To demonstrate the capabilities of this implementation, we developed four proof-of-concept applications for (1) prediction of free energy changes upon mutation; (2) high-resolution structural refinement; (3) protein-protein docking; and (4) assembly of symmetric protein complexes, all in the membrane environment. Preliminary data show that these algorithms can produce meaningful scores and structures. The data also suggest needed improvements to both sampling routines and score functions. Importantly, the applications collectively demonstrate the potential of combining the flexible nature of RosettaMP with the power of Rosetta algorithms to facilitate membrane protein modeling and design. PMID:26325167

  12. On the mode of integration of the thylakoid membrane protein cytochrome b(6) into cytoplasmic membrane of Escherichia coli.

    PubMed

    Króliczewski, Jaroslaw; Gubernator, Beata; Rögner, Matthias; Szczepaniak, Andrzej

    2011-01-01

    In the stroma compartment, several pathways are used for integration/translocation of chloroplast proteins into or across the thylakoid membrane. In this study we investigated the mode of incorporation of the chloroplast-encoded cytochrome b(6) into the bacterial membrane. Cytochrome b(6) naturally comprises of four transmembrane helices (A,B,C,D) and contains two b-type hemes. In the present study, mature cytochrome b(6) or constructed deletion mutants of cytochrome were expressed in E. coli cells. The membrane insertion of cytochrome b(6) in this bacterial model system requires an artificially added presequence that directs the protein to use an E. coli membrane-insertion pathway. This could be accomplished by fusion to maltose-binding protein (MBP) or to the bacterial Sec-dependent signal peptide (SSpelB). The integration of mature cytochrome b(6) into the bacterial cytoplasmic membrane by the Sec pathway has been reported previously by our group (Kroliczewski et al., 2005, Biochemistry, 44: 7570). The results presented here show that cytochrome b(6) devoid of the first helix A can be inserted into the membrane, as can the entire ABCD. On the other hand, the construct devoid of helices A and B is translocated through the membrane into the periplasm without any effective insertion. This suggests the importance of the membrane-anchoring sequences that are likely to be present in only the A and B part, and it is consistent with the results of computational prediction which did not identify any membrane-anchoring sequences for the C or D helices. We also show that the incorporation of hemes into the truncated form of cytochrome b(6) is possible, as long as the B and D helices bearing axial ligands to heme are present. PMID:21725502

  13. Structure of the Membrane Proximal Oxioreductase Domain of Human Steap3, the Dominant Ferrireductase of the Erythroid Transferrin Cycle

    SciTech Connect

    Sendamarai, A.K.; Ohgami, R.S.; Fleming, M.D.; Lawrence, C.M.

    2009-05-27

    The daily production of 200 billion erythrocytes requires 20 mg of iron, accounting for nearly 80% of the iron demand in humans. Thus, erythroid precursor cells possess an efficient mechanism for iron uptake in which iron loaded transferrin (Tf) binds to the transferrin receptor (TfR) at the cell surface. The Tf:TfR complex then enters the endosome via receptor-mediated endocytosis. Upon endosomal acidification, iron is released from Tf, reduced to Fe{sup 2+} by Steap3, and transported across the endosomal membrane by divalent metal iron transporter 1. Steap3, the major ferrireductase in erythrocyte endosomes, is a member of a unique family of reductases. Steap3 is comprised of an N-terminal cytosolic oxidoreductase domain and a C-terminal heme-containing transmembrane domain. Cytosolic NADPH and a flavin are predicted cofactors, but the NADPH/flavin binding domain differs significantly from those in other eukaryotic reductases. Instead, Steap3 shows remarkable, although limited homology to FNO, an archaeal oxidoreductase. We have determined the crystal structure of the human Steap3 oxidoreductase domain in the absence and presence of NADPH. The structure reveals an FNO-like domain with an unexpected dimer interface and substrate binding sites that are well positioned to direct electron transfer from the cytosol to a heme moiety predicted to be fixed within the transmembrane domain. Here, we discuss possible gating mechanisms for electron transfer across the endosomal membrane.

  14. Structure of the membrane proximal oxidoreductase domain of human Steap3, the dominant ferrireductase of the erythroid transferrin cycle

    PubMed Central

    Sendamarai, Anoop K.; Ohgami, Robert S.; Fleming, Mark D.; Lawrence, C. Martin

    2008-01-01

    The daily production of 200 billion erythrocytes requires 20 mg of iron, accounting for nearly 80% of the iron demand in humans. Thus, erythroid precursor cells possess an efficient mechanism for iron uptake in which iron loaded transferrin (Tf) binds to the transferrin receptor (TfR) at the cell surface. The Tf:TfR complex then enters the endosome via receptor-mediated endocytosis. Upon endosomal acidification, iron is released from Tf, reduced to Fe2+ by Steap3, and transported across the endosomal membrane by divalent metal iron transporter 1. Steap3, the major ferrireductase in erythrocyte endosomes, is a member of a unique family of reductases. Steap3 is comprised of an N-terminal cytosolic oxidoreductase domain and a C-terminal heme-containing transmembrane domain. Cytosolic NADPH and a flavin are predicted cofactors, but the NADPH/flavin binding domain differs significantly from those in other eukaryotic reductases. Instead, Steap3 shows remarkable, although limited homology to FNO, an archaeal oxidoreductase. We have determined the crystal structure of the human Steap3 oxidoreductase domain in the absence and presence of NADPH. The structure reveals an FNO-like domain with an unexpected dimer interface and substrate binding sites that are well positioned to direct electron transfer from the cytosol to a heme moiety predicted to be fixed within the transmembrane domain. Here, we discuss possible gating mechanisms for electron transfer across the endosomal membrane. PMID:18495927

  15. A new integrated approach for dye removal from wastewater by polyoxometalates functionalized membranes.

    PubMed

    Yao, Lei; Zhang, Lizhi; Wang, Rong; Chou, Shuren; Dong, ZhiLi

    2016-01-15

    Membrane technique is a promising way for the removal of dyes from wastewater. A unique approach combining both the adsorptive and the catalytic membrane processes was proposed on the basis of a new functionalized membrane. The membrane integrating both the adsorptive and catalytic activities was developed by introducing polyoxometalates (POMs) as an ideal candidate for the membrane functionalization via a novel sol-gel method. A two-step protocol, adsorptive separation and catalytic degradation, was designed for dye removal, realizing an excellent dye rejection with easy and economic membrane regeneration through simply soaking the membrane in a limited volume of dilute oxidant solution. This approach is feasible and versatile owing to the flexible selection of distinct POMs and design of catalytic degradation routes as required. As a result, the current research provides insight into a new methodology of the membrane technique in dye removal applications. PMID:26410275

  16. Multiple Membrane-Cytoplasmic Domain Contacts in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Mediate Regulation of Channel Gating*S⃞

    PubMed Central

    He, Lihua; Aleksandrov, Andrei A.; Serohijos, Adrian W. R.; Hegedüs, Tamás; Aleksandrov, Luba A.; Cui, Liying; Dokholyan, Nikolay V.; Riordan, John R.

    2008-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a unique ATP-binding cassette (ABC) ion channel mutated in patients with cystic fibrosis. The most common mutation, deletion of phenylalanine 508 (ΔF508) and many other disease-associated mutations occur in the nucleotide binding domains (NBD) and the cytoplasmic loops (CL) of the membrane-spanning domains (MSD). A recently constructed computational model of the CFTR three-dimensional structure, supported by experimental data (Serohijos, A. W., Hegedus, T., Aleksandrov, A. A., He, L., Cui, L., Dokholyan, N. V., and Riordan, J. R. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 3256–3261) revealed that several of these mutations including ΔF508 disrupted interfaces between these domains. Here we have used cysteine cross-linking experiments to verify all NBD/CL interfaces predicted by the structural model and observed that their cross-linking has a variety of different effects on channel gating. The interdomain contacts comprise aromatic clusters important for stabilization of the interfaces and also involve the Q-loops and X-loops that are in close proximity to the ATP binding sites. Cross-linking of all domain-swapping contacts between NBDs and MSD cytoplasmic loops in opposite halves of the protein rapidly and reversibly arrest single channel gating while those in the same halves have lesser impact. These results reinforce the idea that mediation of regulatory signals between cytoplasmic- and membrane-integrated domains of the CFTR channel apparently relies on an array of precise but highly dynamic interdomain structural joints. PMID:18658148

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

    PubMed Central

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

    2002-01-01

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

  18. A frequent kinase domain mutation that changes the interaction between PI3K[alpha] and the membrane

    SciTech Connect

    Mandelker, Diana; Gabelli, Sandra B.; Schmidt-Kittler, Oleg; Zhu, Jiuxiang; Cheong, Ian; Huang, Chuan-Hsiang; Kinzler, Kenneth W.; Vogelstein, Bert; Amzel, L. Mario

    2009-12-01

    Mutations in oncogenes often promote tumorigenesis by changing the conformation of the encoded proteins, thereby altering enzymatic activity. The PIK3CA oncogene, which encodes p110{alpha}, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3K{alpha}), is one of the two most frequently mutated oncogenes in human cancers. We report the structure of the most common mutant of p110{alpha} in complex with two interacting domains of its regulatory partner (p85{alpha}), both free and bound to an inhibitor (wortmannin). The N-terminal SH2 (nSH2) domain of p85{alpha} is shown to form a scaffold for the entire enzyme complex, strategically positioned to communicate extrinsic signals from phosphopeptides to three distinct regions of p110{alpha}. Moreover, we found that Arg-1047 points toward the cell membrane, perpendicular to the orientation of His-1047 in the WT enzyme. Surprisingly, two loops of the kinase domain that contact the cell membrane shift conformation in the oncogenic mutant. Biochemical assays revealed that the enzymatic activity of the p110{alpha} His1047Arg mutant is differentially regulated by lipid membrane composition. These structural and biochemical data suggest a previously undescribed mechanism for mutational activation of a kinase that involves perturbation of its interaction with the cellular membrane.

  19. Topological Predictions for Integral Membrane Channel and Carrier Proteins

    PubMed Central

    Abhinay, Reddy; Jaehoon, Cho; Sam, Ling; Vamsee, Reddy; Maksim, Shlykov; Milton, Saier

    2014-01-01

    We evaluated topological predictions for nine different programs, HMMTOP, TMHMM, SVMTOP, DAS, SOSUI, TOPCONS, PHOBIUS, MEMSAT-SVM (hereinafter referred to as MEMSAT), and SPOCTOPUS. These programs were first evaluated using four large topologically well-defined families of secondary transporters, and the three best programs were further evaluated using topologically more diverse families of channels and carriers. In the initial studies, the order of accuracy was: SPOCTOPUS>MEMSAT>HMMTOP>TOPCONS>PHOBIUS>TMHMM>SVMTOP>DAS>S OSUI. Some families, such as the Sugar Porter family (2.A.1.1) of the Major Facilitator Superfamily (MFS; TC# 2.A.1) and the Amino acid/Polyamine/Organocation (APC) Family (TC# 2.A.3), were correctly predicted with high accuracy while others, such as the Mitochondrial Carrier (MC) (TC# 2.A.29) and the K+ transporter (Trk) families (TC# 2.A.38), were predicted with much lower accuracy. For small, topologically homogeneous families, SPOCTOPUS and MEMSAT were generally most reliable, while with large, more diverse superfamilies, HMMTOP often proved to have the greatest prediction accuracy. We next developed a novel program, TM-STATS, that tabulates HMMTOP, SPOCTOPUS or MEMSAT-based topological predictions for any subdivision (class, subclass, superfamily, family, subfamily, or any combination of these) of the Transporter Classification Database (TCDB; www.tcdb.org) and examined the following subclasses: α-type channel proteins (TC subclasses 1.A and 1.E), secreted poreforming toxins (TC subclass 1.C) and secondary carriers (subclass 2.A). Histograms 3 were generated for each of these subclasses, and the results were analyzed according to subclass, family and protein. The results provide an update of topological predictions for integral membrane transport proteins as well as guides for the development of more reliable topological prediction programs, taking family-specific characteristics into account. PMID:24992992

  20. Membrane binding of Escherichia coli RNase E catalytic domain stabilizes protein structure and increases RNA substrate affinity

    PubMed Central

    Murashko, Oleg N.; Kaberdin, Vladimir R.; Lin-Chao, Sue

    2012-01-01

    RNase E plays an essential role in RNA processing and decay and tethers to the cytoplasmic membrane in Escherichia coli; however, the function of this membrane–protein interaction has remained unclear. Here, we establish a mechanistic role for the RNase E–membrane interaction. The reconstituted highly conserved N-terminal fragment of RNase E (NRne, residues 1–499) binds specifically to anionic phospholipids through electrostatic interactions. The membrane-binding specificity of NRne was confirmed using circular dichroism difference spectroscopy; the dissociation constant (Kd) for NRne binding to anionic liposomes was 298 nM. E. coli RNase G and RNase E/G homologs from phylogenetically distant Aquifex aeolicus, Haemophilus influenzae Rd, and Synechocystis sp. were found to be membrane-binding proteins. Electrostatic potentials of NRne and its homologs were found to be conserved, highly positive, and spread over a large surface area encompassing four putative membrane-binding regions identified in the “large” domain (amino acids 1–400, consisting of the RNase H, S1, 5′-sensor, and DNase I subdomains) of E. coli NRne. In vitro cleavage assay using liposome-free and liposome-bound NRne and RNA substrates BR13 and GGG-RNAI showed that NRne membrane binding altered its enzymatic activity. Circular dichroism spectroscopy showed no obvious thermotropic structural changes in membrane-bound NRne between 10 and 60 °C, and membrane-bound NRne retained its normal cleavage activity after cooling. Thus, NRne membrane binding induced changes in secondary protein structure and enzymatic activation by stabilizing the protein-folding state and increasing its binding affinity for its substrate. Our results demonstrate that RNase E–membrane interaction enhances the rate of RNA processing and decay. PMID:22509045

  1. Daptomycin exerts rapid bactericidal activity against Bacillus anthracis without disrupting membrane integrity

    PubMed Central

    Xing, Yu-hua; Wang, Wei; Dai, Su-qin; Liu, Ti-yan; Tan, Jun-jie; Qu, Guo-long; Li, Yu-xia; Ling, Yan; Liu, Gang; Fu, Xue-qi; Chen, Hui-peng

    2014-01-01

    Aim: To examine whether the novel cyclic lipopeptide antibiotic daptomycin could be used to treat anthrax and to study the mechanisms underlying its bactericidal action against Bacillus anthracis. Methods: Spore-forming B anthracis AP422 was tested. MIC values of antibiotics were determined. Cell membrane potential was measured using flow cytometric assays with membrane potential-sensitive fluorescent dyes. Cell membrane integrity was detected using To-Pro-3 iodide staining and transmission electron microscopy. K+ efflux and Na+ influx were measured using the fluorescent probes PBFI and SBFI-AM, respectively. Results: Daptomycin exhibited rapid bactericidal activity against vegetative B anthracis with a MIC value of 0.78 μg/mL, which was comparable to those of ciprofloxacin and penicillin G. Furthermore, daptomycin prevented the germinated spores from growing into vegetative bacteria. Daptomycin concentration-dependently dissipated the membrane potential of B anthracis and caused K+ efflux and Na+ influx without disrupting membrane integrity. In contrast, both ciprofloxacin and penicillin G did not change the membrane potential of vegetative bacteria or spores. Penicillin G disrupted membrane integrity of B anthracis, whereas ciprofloxacin had no such effect. Conclusion: Daptomycin exerts rapid bactericidal action against B anthracis via reducing membrane potential without disrupting membrane integrity. This antibiotic can be used as an alternate therapy for B anthracis infections. PMID:24362329

  2. Binding of the cytoplasmic domain of CD28 to the plasma membrane inhibits Lck recruitment and signaling.

    PubMed

    Dobbins, Jessica; Gagnon, Etienne; Godec, Jernej; Pyrdol, Jason; Vignali, Dario A A; Sharpe, Arlene H; Wucherpfennig, Kai W

    2016-01-01

    The T cell costimulatory receptor CD28 is required for the full activation of naïve T cells and for the development and maintenance of Foxp3(+) regulatory T (Treg) cells. We showed that the cytoplasmic domain of CD28 was bound to the plasma membrane in resting cells and that ligand binding to CD28 resulted in its release. Membrane binding by the CD28 cytoplasmic domain required two clusters of basic amino acid residues, which interacted with the negatively charged inner leaflet of the plasma membrane. These same clusters of basic residues also served as interaction sites for Lck, a Src family kinase critical for CD28 function. This signaling complex was further stabilized by the Lck-mediated phosphorylation of CD28 Tyr(207) and the subsequent binding of the Src homology 2 (SH2) domain of Lck to this phosphorylated tyrosine. Mutation of the basic clusters in the CD28 cytoplasmic domain reduced the recruitment to the CD28-Lck complex of protein kinase Cθ (PKCθ), which serves as a key effector kinase in the CD28 signaling pathway. Consequently, mutation of either a basic cluster or Tyr(207) impaired CD28 function in mice as shown by the reduced thymic differentiation of FoxP3(+) Treg cells. On the basis of these results, we propose a previously undescribed model for the initiation of CD28 signaling. PMID:27460989

  3. Regulation of Renal Organic Anion Transporter 3 (SLC22A8) Expression and Function by the Integrity of Lipid Raft Domains and their Associated Cytoskeleton

    PubMed Central

    Srimaroeng, Chutima; Cecile, Jennifer Perry; Walden, Ramsey; Pritchard, John B.

    2013-01-01

    Background/Aims In humans and rodents, organic anion transporter 3 (Oat3) is highly expressed on the basolateral membrane of renal proximal tubules and mediates the secretion of exogenous and endogenous anions. Regulation of Oat3 expression and function has been observed in both expression system and intact renal epithelia. However, information on the local membrane environment of Oat3 and its role is limited. Lipid raft domains (LRD; cholesterol-rich domains of the plasma membrane) play important roles in membrane protein expression, function and targeting. In the present study, we have examined the role of LRD-rich membranes and their associated cytoskeletal proteins on Oat3 expression and function. Methods LRD-rich membranes were isolated from rat renal cortical tissues and from HEK-293 cells stably expressing human OAT3 (hOAT3) by differential centrifugation with triton X-100 extraction. Western blots were subsequently analyzed to determine protein expression. In addition, the effect of disruption of LRD-rich membranes was examined on functional Oat3 mediated estrone sulfate (ES) transport in rat renal cortical slices. Cytoskeleton disruptors were investigated in both hOAT3 expressing HEK-293 cells and rat renal cortical slices. Results Lipid-enriched membranes from rat renal cortical tissues and hOAT3-expressing HEK-293 cells showed co-expression of rOat3/hOAT3 and several lipid raft-associated proteins, specifically caveolin 1 (Cav1), β-actin and myosin. Moreover, immunohistochemistry in hOAT3-expressing HEK-293 cells demonstrated that these LRD-rich proteins co-localized with hOAT3. Potassium iodide (KI), an inhibitor of protein-cytoskeletal interaction, effectively detached cytoskeleton proteins and hOAT3 from plasma membrane, leading to redistribution of hOAT3 into non-LRD-rich compartments. In addition, inhibition of cytoskeleton integrity and membrane trafficking processes significantly reduced ES uptake mediated by both human and rat Oat3. Cholesterol

  4. Genetic dissection of the outer membrane secretin PulD: are there distinct domains for multimerization and secretion specificity?

    PubMed

    Guilvout, I; Hardie, K R; Sauvonnet, N; Pugsley, A P

    1999-12-01

    Linker and deletion mutagenesis and gene fusions were used to probe the possible domain structure of the dodecameric outer membrane secretin PulD from the pullulanase secretion pathway of Klebsiella oxytoca. Insertions of 24 amino acids close to or within strongly predicted and highly conserved amphipathic beta strands in the C-terminal half of the polypeptide (the beta domain) abolished sodium dodecyl sulfate (SDS)-resistant multimer formation that is characteristic of this protein, whereas insertions elsewhere generally had less dramatic effects on multimer formation. However, the beta domain alone did not form SDS-resistant multimers unless part of the N-terminal region of the protein (the N domain) was produced in trans. All of the insertions except one, close to the C terminus of the protein, abolished function. The N domain alone was highly unstable and did not form SDS-resistant multimers even when the beta domain was present in trans. We conclude that the beta domain is a major determinant of multimer stability and that the N domain contributes to multimer formation. The entire or part of the N domain of PulD could be replaced by the corresponding region of the OutD secretin from the pectate lyase secretion pathway of Erwinia chrysanthemi without abolishing pullulanase secretion. This suggests that the N domain of PulD is not involved in substrate recognition, contrary to the role proposed for the N domain of OutD, which binds specifically to pectate lyase secreted by E. chrysanthemi (V. E. Shevchik, J. Robert-Badouy, and G. Condemine, EMBO J. 16:3007-3016, 1997). PMID:10572123

  5. Large structure rearrangement of colicin ia channel domain after membrane binding from 2D 13C spin diffusion NMR.

    PubMed

    Luo, Wenbin; Yao, Xiaolan; Hong, Mei

    2005-05-01

    One of the main mechanisms of membrane protein folding is by spontaneous insertion into the lipid bilayer from the aqueous environment. The bacterial toxin, colicin Ia, is one such protein. To shed light on the conformational changes involved in this dramatic transfer from the polar to the hydrophobic milieu, we carried out 2D magic-angle spinning (13)C NMR experiments on the water-soluble and membrane-bound states of the channel-forming domain of colicin Ia. Proton-driven (13)C spin diffusion spectra of selectively (13)C-labeled protein show unequivocal attenuation of cross-peaks after membrane binding. This attenuation can be assigned to distance increases but not reduction of the diffusion coefficient. Analysis of the statistics of the interhelical and intrahelical (13)C-(13)C distances in the soluble protein structure indicates that the observed cross-peak reduction is well correlated with a high percentage of short interhelical contacts in the soluble protein. This suggests that colicin Ia channel domain becomes open and extended upon membrane binding, thus lengthening interhelical distances. In comparison, cross-peaks with similar intensities between the two states are dominated by intrahelical contacts in the soluble state. This suggests that the membrane-bound structure of colicin Ia channel domain may be described as a "molten globule", in which the helical secondary structure is retained while the tertiary structure is unfolded. This study demonstrates that (13)C spin diffusion NMR is a valuable tool for obtaining qualitative long-range distance constraints on membrane protein folding. PMID:15853348

  6. Mechanisms of integral membrane protein insertion and folding

    PubMed Central

    2014-01-01

    The biogenesis, folding, and structure of α-helical membrane proteins (MPs) are important to understand because they underlie virtually all physiological processes in cells including key metabolic pathways, such as the respiratory chain and the photosystems, and the transport of solutes and signals across membranes. Nearly all MPs require translocons—often referred to as protein-conducting channels—for proper insertion into their target membrane. Remarkable progress toward understanding the structure and functioning of translocons has been made during the past decade. Here we review and assess this progress critically. All available evidence indicates that MPs are equilibrium structures that achieve their final structural states by folding along thermodynamically controlled pathways. The main challenge for cells is the targeting and membrane insertion of highly hydrophobic amino acid sequences. Targeting and insertion are managed in cells principally by interactions between ribosomes and membrane-embedded translocons. Our review examines the biophysical and biological boundaries of membrane protein insertion and the folding of polytopic membrane proteins in vivo. A theme of the review is the under-appreciated role of basic thermodynamic principles in MP folding and assembly. Thermodynamics not only dictates the final folded structure, it is the driving force for the evolution of the ribosome-translocon system of assembly. We conclude the review with a perspective suggesting a new view of translocon-guided MP insertion. PMID:25277655

  7. Perspectives of solution NMR spectroscopy for structural and functional studies of integral membrane proteins

    NASA Astrophysics Data System (ADS)

    Reckel, Sina; Hiller, Sebastian

    2013-04-01

    This article discusses future perspectives of solution NMR spectroscopy to study structures and functions of integral membrane proteins at atomic resolution, based on a review of recent progress in this area. Several selected examples of structure determinations, as well as functional studies of integral membrane proteins are highlighted. We expect NMR spectroscopy to make future key scientific contributions to understanding membrane protein function, in particular for large membrane protein systems with known three-dimensional structure. Such situations can benefit from the fact that functional NMR studies have substantially less limitations by molecular size than a full de novo structure determination. Therefore, the general potential for NMR spectroscopy to solve biologic key questions associated with integral membrane proteins is very promising.

  8. The hypo-osmotic swelling test for evaluation of sperm membrane integrity.

    PubMed

    Ramu, Sivakumar; Jeyendran, Rajasingam S

    2013-01-01

    A functional membrane is requisite for the fertilizing ability of spermatozoa, as it plays an integral role in sperm capacitation, acrosome reaction, and binding of the spermatozoon to the egg surface. The hypo-osmotic swelling (HOS) test evaluates the functional integrity of the sperm's plasma membrane and also serves as a useful indicator of fertility potential of sperm. The HOS test predicts membrane integrity by determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its environment. Influx of the fluid due to hypo-osmotic stress causes the sperm tail to coil and balloon or "swell." A higher percentage of swollen sperm indicates the presence of sperm having a functional and intact plasma membrane. Here, we present the detailed protocol for performing the HOS test and explain the results for interpretation. PMID:22992900

  9. Cardiac muscle cell cytoskeletal protein 4.1: analysis of transcripts and subcellular location--relevance to membrane integrity, microstructure, and possible role in heart failure.

    PubMed

    Taylor-Harris, Pamela M; Keating, Lisa A; Maggs, Alison M; Phillips, Gareth W; Birks, Emma J; Franklin, Rodney C G; Yacoub, Magdi H; Baines, Anthony J; Pinder, Jennifer C

    2005-03-01

    The spectrin-based cytoskeleton assembly has emerged as a major player in heart functioning; however, cardiac protein 4.1, a key constituent, is uncharacterized. Protein 4.1 evolved to protect cell membranes against mechanical stresses and to organize membrane microstructure. 4.1 Proteins are multifunctional and, among other activities, link integral/signaling proteins on the plasma and internal membranes with the spectrin-based cytoskeleton. Four genes, EPB41, EPB41L1, EPB41L2, and EPB41L3 encode proteins 4.1R, 4.1N, 4.1G, and 4.1B, respectively. All are extensively spliced. Different isoforms are expressed according to tissue and developmental state, individual function being controlled through inclusion/exclusion of interactive domains. We have defined mouse and human cardiac 4.1 transcripts; other than 4. 1B in humans, all genes show activity. Cardiac transcripts constitutively include conserved FERM and C-terminal domains; both interact with membrane-bound signaling/transport/cell adhesion molecules. Variable splicing within and adjacent to the central spectrin/actin-binding domain enables regulation of cytoskeleton-binding activity. A novel heart-specific exon occurs in human 4.1G, but not in mouse. Immunofluorescence reveals 4.1 staining within mouse cardiomyocytes; thus, both at the plasma membrane and, interdigitated with sarcomeric myosin, across myofibrils in regions close to the sarcoplasmic reticulum. These are all regions to which spectrin locates. 4.1R in human heart shows similar distribution; however, there is limited plasma membrane staining. We conclude that cardiac 4.1s are highly regulated in their ability to crosslink plasma/integral cell membranes with the spectrin-actin cytoskeleton. We speculate that over the repetitive cycles of heart muscle contraction and relaxation, 4.1s are likely to locate, support, and coordinate functioning of key membrane-bound macromolecular assemblies. PMID:15834631

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

    PubMed Central

    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.

    2015-01-01

    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. PMID:25838348

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

    PubMed Central

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

    2015-01-01

    The C-terminal transmembrane domain (TMD) of viral fusion proteins such as HIV gp41 and influenza hemagglutinin (HA) is traditionally viewed as a passive α-helical anchor of the protein to the virus envelope during its merger with the cell membrane. The conformation, dynamics, and lipid interaction of these fusion protein TMDs have so far eluded high-resolution structure characterization because of their highly hydrophobic nature. Using magic-angle-spinning solid-state NMR spectroscopy, we show that the TMD of the parainfluenza virus 5 (PIV5) fusion protein adopts lipid-dependent conformations and interactions with the membrane and water. In phosphatidylcholine (PC) and phosphatidylglycerol (PG) membranes, the TMD is predominantly α-helical, but in phosphatidylethanolamine (PE) membranes, the TMD changes significantly to the β-strand conformation. Measured order parameters indicate that the strand segments are immobilized and thus oligomerized. 31P NMR spectra and small-angle X-ray scattering (SAXS) data show that this β-strand–rich conformation converts the PE membrane to a bicontinuous cubic phase, which is rich in negative Gaussian curvature that is characteristic of hemifusion intermediates and fusion pores. 1H-31P 2D correlation spectra and 2H spectra show that the PE membrane with or without the TMD is much less hydrated than PC and PG membranes, suggesting that the TMD works with the natural dehydration tendency of PE to facilitate membrane merger. These results suggest a new viral-fusion model in which the TMD actively promotes membrane topological changes during fusion using the β-strand as the fusogenic conformation. PMID:26283363

  12. Partial structural characterization of the cytoplasmic domain of the erythrocyte membrane protein, band 3.

    PubMed

    Appell, K C; Low, P S

    1981-11-10

    The cytoplasmic domain of band 3 was released from spectrin-depleted, acetic acid-stripped erythrocyte membrane vesicles by mild chymotryptic digestion. After purification by ion exchange and gel filtration chromatography, the fragment preparation was found to be greater than 90% pure on polyacrylamide disc gels in the presence of 0.2% sodium dodecyl sulfate. The subunit Mr from the electrophoretic procedure was estimated at approximately 40,000. The isolated cytoplasmic fragment ws judged to be a dimer, since (i) the unmodified fragment and its disulfide-cross-linked (dimeric) counterpart eluted in the same peak fraction from a Sephacryl S-200 gel filtration column, and (ii) the sedimentation velocity molecular weight of the native fragment was calculated to be approximately 95,000. No evidence of either larger or smaller aggregates was obtained. The frictional ratio of the fragment was measured at 1.6, suggesting a highly elongated morphology. The circular dichroism spectrum of the fragment corresponded to approximately 37% alpha helix. Titration of the cytoplasmic fragment over the physiological pH range gave rise to a reversible 2-fold increase in the intrinsic fluorescence quantum yield (lambda ex, 290 nm; lambda em, 335 nm) between pH 6 and 9. Computer analysis of the data yielded a temperature-dependent apparent pKa of 7.8 at 37 degrees C and 8.1 at 20 degrees C, both with Hill coefficients less than or equal to 1. Calorimetric experiments revealed a similar sensitivity to pH, where the denaturation temperature of the fragment titrated from 74 degrees C at pH 6 to 59 degrees C at pH 8.5, with an apparent pKa of 7.3 and a Hill coefficient less than 1. The enthalpies and widths at half-height of the transitions were also exquisitely sensitive to pH. The fluorescence and calorimetric data could all be described by the titration of a single ionizable group of apparent pKa of 7.8 at 37 degrees C and delta pKa/degrees C of -0.018. The ionization of this critical

  13. Alteration of membrane integrity by delta1-tetrahydrocannabinol.

    PubMed

    Laurent, B; Roy, P E

    1975-07-01

    Delta1-tetrahydrocannabinol was found to be a potent inhibitor of some membrane-bound enzymes, such as Mg-ATPase, Na-K-ATPase and acetylcholinesterase. At a given concentration, the degree of inhibition varied for each enzyme; the inhibition was more pronounced for the enzymes that are parts of the membranes. As the kinetic parameters of these enzymes are functions of the membrane composition and organization, these parameters were studied in vitro in the presence of THC. Although the Mg-ATPase was inhibited by THC, there was no change in the allosteric behaviour of the enzyme, indicating that the alterations caused by THC did not affect the enzymatic structure. The Na-K-ATPase and acetylcholinesterase had a different allosteric behaviour as compared to controls; these modifications were like the alterations caused by the decrease in membrane fluidity. These results suggest the fact that THC is incorporated in the membranes and causes alterations in the physical organization of the membranes. PMID:126214

  14. Analysis of the membrane-interacting domain of the sea urchin sperm adhesive protein bindin

    SciTech Connect

    Kennedy, L.; DeAngelis, P.L.; Glabe, C.G. )

    1989-11-14

    The authors have investigated the domain of the bindin polypeptide the selectively associates with gel-phase phospholipid vesicles. They found that small trypsin fragments of bindin retain the ability to selectively associate with gel-phase vesicles. The primary amino acid sequence of bindin suggests that these peptides are derived from the central portion of the polypeptide between residues 77 and 126, which is the most hydrophobic region of bindin. They have also employed 3-(trifluoromethyl)-3-(m-({sup 125}I)iodophenyl)diazirine (TID) and novel, radioiodinated, photoactivatable derivatives of the polar head group of phosphatidylethanolamine (ASD-PE and ASA-PE) to identify membrane-associated polypeptide segments after the transfer of radiolabel from the probe to the bindin polypeptide. After photolysis, bindin was selectively labeled only from probes incorporated in gel-phase vesicles. The labeling of bindin was much more efficient from the head group probes ASA-PE and ASD-PE (8 and 2% of the total label, respectively) in comparison to the hydrophobic probe TID (less than 0.02% of the total label), suggesting that bindin is localized within the polar part of the bilayer. Protease mapping experiments with V8 protease, trypsin, and endoprotease Lys-C suggest that some of the probe label is distributed along the amino-terminal portion of bindin between residues 1 and 76 and the rest of the label is restricted to the segments between residues 77 and 126 which also selectively bind to gel-phase vesicles. The carboxyl-terminal portion of bindin residues 127 and 236 is not labeled.

  15. Efficient ethanol recovery from fermentation broths with integrated distillation-membrane process

    EPA Science Inventory

    The energy demand of distillation-molecular sieve systems for ethanol recovery/dehydration can be significant, particularly for dilute solutions. An alternative process integrating vapor stripping (like a beer still) with vapor compression and a vapor permeation membrane separati...

  16. Functional characterization of the trans-membrane domain interactions of the Sec61 protein translocation complex beta-subunit

    PubMed Central

    Zhao, Xueqiang; Jäntti, Jussi

    2009-01-01

    Background In eukaryotic cells co- and post-translational protein translocation is mediated by the trimeric Sec61 complex. Currently, the role of the Sec61 complex β-subunit in protein translocation is poorly understood. We have shown previously that in Saccharomyces cerevisiae the trans-membrane domain alone is sufficient for the function of the β-subunit Sbh1p in co-translational protein translocation. In addition, Sbh1p co-purifies not only with the protein translocation channel subunits Sec61p and Sss1p, but also with the reticulon family protein Rtn1p. Results We used random mutagenesis to generate novel Sbh1p mutants in order to functionally map the Sbh1p trans-membrane domain. These mutants were analyzed for their interactions with Sec61p and how they support co-translational protein translocation. The distribution of mutations identifies one side of the Sbh1p trans-membrane domain α-helix that is involved in interactions with Sec61p and that is important for Sbh1p function in protein translocation. At the same time, these mutations do not affect Sbh1p interaction with Rtn1p. Furthermore we show that Sbh1p is found in protein complexes containing not only Rtn1p, but also the two other reticulon-like proteins Rtn2p and Yop1p. Conclusion Our results identify functionally important amino acids in the Sbh1p trans-membrane domain. In addition, our results provide additional support for the involvement of Sec61β in processes unlinked to protein translocation. PMID:19857245

  17. PD-L1 Antibodies to Its Cytoplasmic Domain Most Clearly Delineate Cell Membranes in Immunohistochemical Staining of Tumor Cells.

    PubMed

    Mahoney, Kathleen M; Sun, Heather; Liao, Xiaoyun; Hua, Ping; Callea, Marcella; Greenfield, Edward A; Hodi, F Stephen; Sharpe, Arlene H; Signoretti, Sabina; Rodig, Scott J; Freeman, Gordon J

    2015-12-01

    Blocking the programmed death-1 (PD-1) pathway has clinical benefit in metastatic cancer and has led to the approval of the mAbs pembrolizumab and nivolumab to treat melanoma and nivolumab for non-small cell lung cancer. Expression of PD-L1 on the cell surface of either tumor cells or infiltrating immune cells is associated with a higher likelihood of response to PD-1 blockade in multiple studies. Most mAbs to PD-L1 in use are directed to its extracellular domain and immunohistochemically stain tumor tissue with a mixture of cytoplasmic and membrane staining. Cytoplasmic staining obscures the interpretation of a positive reaction on the tumor cell membrane, and thus affects the accuracy of PD-L1 scoring systems. We developed a mAb to the cytoplasmic domain of PD-L1, 405.9A11 (9A11), which is both more selective for membranous PD-L1 and more sensitive in IHC and Western blotting, compared with previous mAbs specific for the PD-L1 extracellular domain. Here, we compare immunohistochemical staining patterns of PD-L1 expression in five types of tumors, using five PD-L1 mAbs: 9A11, 7G11, and three commercially available mAbs. We demonstrate that 9A11, as well as two other cytoplasmic domain-specific mAbs, E1L3N and SP142, can clearly delineate the membrane of PD-L1-positive cells in formalin-fixed paraffin-embedded tissue and facilitate interpretation of staining results. PMID:26546452

  18. Monitoring the size and lateral dynamics of ErbB1 enriched membrane domains through live cell plasmon coupling microscopy.

    PubMed

    Rong, Guoxin; Reinhard, Björn M

    2012-01-01

    To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60-250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of D = |0.0054±0.0064| µm(2)/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions. PMID:22470534

  19. Monitoring the Size and Lateral Dynamics of ErbB1 Enriched Membrane Domains through Live Cell Plasmon Coupling Microscopy

    PubMed Central

    Rong, Guoxin; Reinhard, Björn M.

    2012-01-01

    To illuminate the role of the spatial organization of the epidermal growth factor receptor (ErbB1) in signal transduction quantitative information about the receptor topography on the cell surface, ideally on living cells and in real time, are required. We demonstrate that plasmon coupling microscopy (PCM) enables to detect, size, and track individual membrane domains enriched in ErbB1 with high temporal resolution. We used a dendrimer enhanced labeling strategy to label ErbB1 receptors on epidermoid carcinoma cells (A431) with 60 nm Au nanoparticle (NP) immunolabels under physiological conditions at 37°C. The statistical analysis of the spatial NP distribution on the cell surface in the scanning electron microscope (SEM) confirmed a clustering of the NP labels consistent with a heterogeneous distribution of ErbB1 in the plasma membrane. Spectral shifts in the scattering response of clustered NPs facilitated the detection and sizing of individual NP clusters on living cells in solution in an optical microscope. We tracked the lateral diffusion of individual clusters at a frame rate of 200 frames/s while simultaneously monitoring the configurational dynamics of the clusters. Structural information about the NP clusters in their membrane confinements were obtained through analysis of the electromagnetic coupling of the co-confined NP labels through polarization resolved PCM. Our studies show that the ErbB1 receptor is enriched in membrane domains with typical diameters in the range between 60–250 nm. These membrane domains exhibit a slow lateral diffusion with a diffusion coefficient of  = |0.0054±0.0064| µm2/s, which is almost an order of magnitude slower than the mean diffusion coefficient of individual NP tagged ErbB1 receptors under identical conditions. PMID:22470534

  20. Integrating membrane filtration into bioelectrochemical systems as next generation energy-efficient wastewater treatment technologies for water reclamation: A review.

    PubMed

    Yuan, Heyang; He, Zhen

    2015-11-01

    Bioelectrochemical systems (BES) represent an energy-efficient approach for wastewater treatment, but the effluent still requires further treatment for direct discharge or reuse. Integrating membrane filtration in BES can achieve high-quality effluents with additional benefits. Three types of filtration membranes, dynamic membrane, ultrafiltration membrane and forward osmosis membrane that are grouped based on pore size, have been studied for integration in BES. The integration can be accomplished either in an internal or an external configuration. In an internal configuration, membranes can act as a separator between the electrodes, or be immersed in the anode/cathode chamber as a filtration component. The external configuration allows BES and membrane module to be operated independently. Given much progress and interest in the integration of membrane filtration into BES, this paper has reviewed the past studies, described various integration methods, discussed the advantages and limitations of each integration, and presented challenges for future development. PMID:26026232

  1. Spin chain from membrane and the Neumann-Rosochatius integrable system

    SciTech Connect

    Bozhilov, P.

    2007-11-15

    We find membrane configurations in AdS{sub 4}xS{sup 7}, which correspond to the continuous limit of the SU(2) integrable spin chain, considered as a limit of the SU(3) spin chain, arising in N=4 SYM in four dimensions, dual to strings in AdS{sub 5}xS{sup 5}. We also discuss the relationship with the Neumann-Rosochatius integrable system at the level of Lagrangians, comparing the string and membrane cases.

  2. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking.

    PubMed

    Wu, Hsiao-Mei; Lin, Ying-Hsiu; Yen, Tzu-Chi; Hsieh, Chia-Lung

    2016-01-01

    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes. PMID:26861908

  3. Conductivity Scaling Relationships of Nanostructured Membranes based on Hydrated Protic Polymerized Ionic Liquids: Effect of Domain Spacing

    NASA Astrophysics Data System (ADS)

    Sanoja, Gabriel; Popere, Bhooshan; Beckingham, Bryan; Evans, Christopher; Lynd, Nathaniel; Segalman, Rachel

    Elucidating the relationship between chemical structure, morphology, and ionic conductivity is essential for designing novel materials for electrochemical applications. In this work, the effect of lamellar domain spacing (d) on ionic conductivity (σ) is investigated for a model system of hydrated block copolymer based on a protic polymerized ionic liquid. We present a strategy that allows for the synthesis of a well-defined series of narrowly dispersed PS- b - PIL with constant volume fraction of ionic liquid moieties (fIL ~ 0.39). These materials self-assemble into ordered lamellar morphologies with variable domain spacing (23-59 nm) as demonstrated by SAXS. PS- b - PIL membranes exhibit ionic conductivities above 10-4 S/cm at room temperature, which are independent of domain spacing. The conductivity scaling relationship demonstrated in this work suggests that a mechanically robust membrane can be designed without compromising its ability to transport ions. In addition, PIL-based membranes exhibit lower water uptake (λ = 10) in comparison with many proton-conducting systems reported elsewhere. The low water content of these materials makes them promising candidates for solar-fuels electrochemical devices.

  4. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

    PubMed Central

    Wu, Hsiao-Mei; Lin, Ying-Hsiu; Yen, Tzu-Chi; Hsieh, Chia-Lung

    2016-01-01

    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes. PMID:26861908

  5. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking

    NASA Astrophysics Data System (ADS)

    Wu, Hsiao-Mei; Lin, Ying-Hsiu; Yen, Tzu-Chi; Hsieh, Chia-Lung

    2016-02-01

    Lipid rafts are membrane nanodomains that facilitate important cell functions. Despite recent advances in identifying the biological significance of rafts, nature and regulation mechanism of rafts are largely unknown due to the difficulty of resolving dynamic molecular interaction of rafts at the nanoscale. Here, we investigate organization and single-molecule dynamics of rafts by monitoring lateral diffusion of single molecules in raft-containing reconstituted membranes supported on mica substrates. Using high-speed interferometric scattering (iSCAT) optical microscopy and small gold nanoparticles as labels, motion of single lipids is recorded via single-particle tracking (SPT) with nanometer spatial precision and microsecond temporal resolution. Processes of single molecules partitioning into and escaping from the raft-mimetic liquid-ordered (Lo) domains are directly visualized in a continuous manner with unprecedented clarity. Importantly, we observe subdiffusion of saturated lipids in the Lo domain in microsecond timescale, indicating the nanoscopic heterogeneous molecular arrangement of the Lo domain. Further analysis of the diffusion trajectory shows the presence of nano-subdomains of the Lo phase, as small as 10 nm, which transiently trap the lipids. Our results provide the first experimental evidence of non-uniform molecular organization of the Lo phase, giving a new view of how rafts recruit and confine molecules in cell membranes.

  6. Essential regions in the membrane domain of bacterial complex I (NDH-1): the machinery for proton translocation.

    PubMed

    Sato, Motoaki; Torres-Bacete, Jesus; Sinha, Prem Kumar; Matsuno-Yagi, Akemi; Yagi, Takao

    2014-08-01

    The proton-translocating NADH-quinone oxidoreductase (complex I/NDH-1) is the first and largest enzyme of the respiratory chain which has a central role in cellular energy production and is implicated in many human neurodegenerative diseases and aging. It is believed that the peripheral domain of complex I/NDH-1 transfers the electron from NADH to Quinone (Q) and the redox energy couples the proton translocation in the membrane domain. To investigate the mechanism of the proton translocation, in a series of works we have systematically studied all membrane subunits in the Escherichia coli NDH-1 by site-directed mutagenesis. In this mini-review, we have summarized our strategy and results of the mutagenesis by depicting residues essential for proton translocation, along with those for subunit connection. It is suggested that clues to understanding the driving forces of proton translocation lie in the similarities and differences of the membrane subunits, highlighting the communication of essential charged residues among the subunits. A possible proton translocation mechanism with all membrane subunits operating in unison is described. PMID:24973951

  7. Solution NMR and X-ray Crystal Structures of Membrane-associated Lipoprotein-17 Domain Reveal a Novel Fold

    SciTech Connect

    R Mani; S Vorobiev; G Swapna; H Neely; H Janjua; C Ciccosanti; D Xiao; J Hunt; G Montelione; et al.

    2011-12-31

    The conserved Lipoprotein-17 domain of membrane-associated protein Q9PRA0{_}UREPA from Ureaplasma parvum was selected for structure determination by the Northeast Structural Genomics Consortium, as part of the Protein Structure Initiative's program on structure-function analysis of protein domains from large domain sequence families lacking structural representatives. The 100-residue Lipoprotein-17 domain is a 'domain of unknown function' (DUF) that is a member of Pfam protein family PF04200, a large domain family for which no members have characterized biochemical functions. The three-dimensional structure of the Lipoprotein-17 domain of protein Q9PRA0{_}UREPA was determined by both solution NMR and by X-ray crystallography at 2.5 {angstrom}. The two structures are in good agreement with each other. The domain structure features three {alpha}-helices, {alpha}1 through {alpha}3, and five {beta}-strands. Strands {beta}1/{beta}2, {beta}3/{beta}4, {beta}4/{beta}5 are anti-parallel to each other. Strands {beta}1 and {beta}2 are orthogonal to strands {beta}3, {beta}4, {beta}5, while helix {alpha}3 is formed between the strands {beta}3 and {beta}4. One-turn helix {alpha}2 is formed between the strands {beta}1 and {beta}2, while helix {alpha}1 occurs in the N-terminal polypeptide segment. Searches of the Protein Data Bank do not identify any other protein with significant structural similarity to Lipoprotein-17 domain of Q9PRA0{_}UREPA, indicating that it is a novel protein fold.

  8. Poloxamer-188 and citicoline provide neuronal membrane integrity and protect membrane stability in cortical spreading depression.

    PubMed

    Yıldırım, Timur; Eylen, Alpaslan; Lule, Sevda; Erdener, Sefik Evren; Vural, Atay; Karatas, Hulya; Ozveren, Mehmet Faik; Dalkara, Turgay; Gursoy-Ozdemir, Yasemin

    2015-01-01

    Under pathological conditions such as brain trauma, subarachnoid hemorrhage and stroke, cortical spreading depression (CSD) or peri-infarct depolarizations contribute to brain damage in animal models of neurological disorders as well as in human neurological diseases. CSD causes transient megachannel opening on the neuronal membrane, which may compromise neuronal survival under pathological conditions. Poloxamer-188 (P-188) and citicoline are neuroprotectants with membrane sealing properties. The aim of this study is to investigate the effect of P-188 and citicoline on the neuronal megachannel opening induced by CSD in the mouse brain. We have monitored megachannel opening with propidium iodide, a membrane impermeable fluorescent dye and, demonstrate that P-188 and citicoline strikingly decreased CSD-induced neuronal PI influx in cortex and hippocampal dentate gyrus. Therefore, these agents may be providing neuroprotection by blocking megachannel opening, which may be related to their membrane sealing action and warrant further investigation for treatment of traumatic brain injury and ischemic stroke. PMID:25340256

  9. Integrated membrane systems for gas separation in biotechnology: potential and prospects.

    PubMed

    Teplyakov, V; Sostina, E; Beckman, I; Netrusov, A

    1996-09-01

    Integrated non-porous membrane systems were applied for microbial combustible gas separation processes. Methane/CO2 mixtures of various concentrations from methane fermentation processes (biogas) were separated using a membrane-separation complex of permabsorber type into individual components of technical grade (more than 95% purity). In experiments with three-component mixtures, using a selective membrane valve with various liquid carriers, all the gases of interest (H2, CH4 and CO2) were obtained at greater than 90% purity in one separation step. The perspectives for the further application of non-porous membrane separating devices for various gaseous mixtures from different microbial processes are discussed. PMID:24415378

  10. Correlation study of microalgae carbonation in membrane integrated photobioreactor

    NASA Astrophysics Data System (ADS)

    Suali, E.; Sarbatly, R.; Shaleh, S. R. M.; Lahin, F. A.; Anisuzzaman, S. M.

    2016-06-01

    Microalgae ability to utilise CO2 higher compared to terrestrial plant making it suitable for biomass production and as CO2 utiliser. This could be one of many ways to preserve a safer and healthier environment with less air pollutant. For study purposes, CO2 usually transported to microalgae culture broth with the aid of membrane technology to prevent formation of large bubble and to accelerate the carbonation of microalgal media. However, membrane susceptible to accumulation of CO2, which can cause extreme acidic to microalgal media. This prevents microalgae to assimilate CO2. Thus, this study proposes correlations to prevent the extreme acidic: which represents the relationship of: (1) CO2 inlet and accumulation, (2) CO2 inlet and CO2 at the membrane-liquid interphase and (3) CO2 inlet and CO2 solubility in the media. The correlations were successfully validated with a deviation of less than 20% compared to the theoretical value.

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

    PubMed

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

    2016-01-15

    The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, but no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. PMID:26655244

  12. RICH-1 has a BIN/Amphiphysin/Rvsp domain responsible for binding to membrane lipids and tubulation of liposomes.

    PubMed

    Richnau, Ninna; Fransson, Asa; Farsad, Khashayar; Aspenström, Pontus

    2004-07-30

    RhoGAP interacting with CIP4 homologs-1 (RICH-1) was previously found in a yeast two-hybrid screen for proteins interacting with the SH3 domain of the Cdc42-interacting protein 4 (CIP4). RICH-1 was shown to be a RhoGAP for Cdc42 and Rac. In this study, we show that the BIN/Amphiphysin/Rvsp (BAR) domain in RICH-1 confers binding to membrane lipids, and has the potential to deform spherical liposomes into tubes. In accordance with previous findings for the BAR domains in endophilin and amphiphysin, RICH-1-induced tubes appeared striated. We propose that these striated structures are formed by oligomerization of RICH-1 through a putative coiled-coil region within the BAR domain. In support of this notion, we show that RICH-1 forms oligomers in the presence of the chemical cross-linker BS3. These results point to an involvement of RICH-1 in membrane deformation events. PMID:15240152

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

    PubMed Central

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

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.11897.001 PMID:26714107

  14. Cooperative Recruitment of Dynamin and BIN/Amphiphysin/Rvs (BAR) Domain-containing Proteins Leads to GTP-dependent Membrane Scission*♦

    PubMed Central

    Meinecke, Michael; Boucrot, Emmanuel; Camdere, Gamze; Hon, Wai-Ching; Mittal, Rohit; McMahon, Harvey T.

    2013-01-01

    Dynamin mediates various membrane fission events, including the scission of clathrin-coated vesicles. Here, we provide direct evidence for cooperative membrane recruitment of dynamin with the BIN/amphiphysin/Rvs (BAR) proteins, endophilin and amphiphysin. Surprisingly, endophilin and amphiphysin recruitment to membranes was also dependent on binding to dynamin due to auto-inhibition of BAR-membrane interactions. Consistent with reciprocal recruitment in vitro, dynamin recruitment to the plasma membrane in cells was strongly reduced by concomitant depletion of endophilin and amphiphysin, and conversely, depletion of dynamin dramatically reduced the recruitment of endophilin. In addition, amphiphysin depletion was observed to severely inhibit clathrin-mediated endocytosis. Furthermore, GTP-dependent membrane scission by dynamin was dramatically elevated by BAR domain proteins. Thus, BAR domain proteins and dynamin act in synergy in membrane recruitment and GTP-dependent vesicle scission. PMID:23297414

  15. High quality single crystal Ge nano-membranes for opto-electronic integrated circuitry

    SciTech Connect

    Shah, V. A. Gammon, P. M.; Rhead, S. D.; Halpin, J. E.; Trushkevych, O.; Wilson, N. R.; Myronov, M.; Edwards, R. S.; Patchett, D. H.; Allred, P. S.; Prest, M. J.; Whall, T. E.; Parker, E. H. C.; Leadley, D. R.; Chávez-Ángel, E.; Shchepetov, A.; Prunnila, M.; Kachkanov, V.; Dolbnya, I. P.; Reparaz, J. S.; and others

    2014-04-14

    A thin, flat, and single crystal germanium membrane would be an ideal platform on which to mount sensors or integrate photonic and electronic devices, using standard silicon processing technology. We present a fabrication technique compatible with integrated-circuit wafer scale processing to produce membranes of thickness between 60 nm and 800 nm, with large areas of up to 3.5 mm{sup 2}. We show how the optical properties change with thickness, including appearance of Fabry-Pérot type interference in thin membranes. The membranes have low Q-factors, which allow the platforms to counteract distortion during agitation and movement. Finally, we report on the physical characteristics showing sub-nm roughness and a homogenous strain profile throughout the freestanding layer, making the single crystal Ge membrane an excellent platform for further epitaxial growth or deposition of materials.

  16. A subset of bacterial inner membrane proteins integrated by the twin-arginine translocase.

    PubMed

    Hatzixanthis, Kostas; Palmer, Tracy; Sargent, Frank

    2003-09-01

    A group of bacterial exported proteins are synthesized with N-terminal signal peptides containing a SRRxFLK 'twin-arginine' amino acid motif. Proteins bearing twin-arginine signal peptides are targeted post-translationally to the twin-arginine translocation (Tat) system which transports folded substrates across the inner membrane. In Escherichia coli, most integral inner membrane proteins are assembled by a co-translational process directed by SRP/FtsY, the SecYEG translocase, and YidC. In this work we define a novel class of integral membrane proteins assembled by a Tat-dependent mechanism. We show that at least five E. coli Tat substrate proteins contain hydrophobic C-terminal transmembrane helices (or 'C-tails'). Fusions between the identified transmembrane C-tails and the exclusively Tat-dependent reporter proteins TorA and SufI render the resultant chimeras membrane-bound. Export-linked signal peptide processing and membrane integration of the chimeras is shown to be both Tat-dependent and YidC-independent. It is proposed that the mechanism of membrane integration of proteins by the Tat system is fundamentally distinct from that employed for other bacterial inner membrane proteins. PMID:12940994

  17. Monitoring Protein-Protein Interactions between the Mammalian Integral Membrane Transporters and PDZ-interacting Partners Using a Modified Split-ubiquitin Membrane Yeast Two-hybrid System*S⃞

    PubMed Central

    Gisler, Serge M.; Kittanakom, Saranya; Fuster, Daniel; Wong, Victoria; Bertic, Mia; Radanovic, Tamara; Hall, Randy A.; Murer, Heini; Biber, Jürg; Markovich, Daniel; Moe, Orson W.; Stagljar, Igor

    2008-01-01

    PDZ-binding motifs are found in the C-terminal tails of numerous integral membrane proteins where they mediate specific protein-protein interactions by binding to PDZ-containing proteins. Conventional yeast two-hybrid screens have been used to probe protein-protein interactions of these soluble C termini. However, to date no in vivo technology has been available to study interactions between the full-length integral membrane proteins and their cognate PDZ-interacting partners. We previously developed a split-ubiquitin membrane yeast two-hybrid (MYTH) system to test interactions between such integral membrane proteins by using a transcriptional output based on cleavage of a transcription factor from the C terminus of membrane-inserted baits. Here we modified MYTH to permit detection of C-terminal PDZ domain interactions by redirecting the transcription factor moiety from the C to the N terminus of a given integral membrane protein thus liberating their native C termini. We successfully applied this “MYTH 2.0” system to five different mammalian full-length renal transporters and identified novel PDZ domain-containing partners of the phosphate (NaPi-IIa) and sulfate (NaS1) transporters that would have otherwise not been detectable. Furthermore this assay was applied to locate the PDZ-binding domain on the NaS1 protein. We showed that the PDZ-binding domain for PDZK1 on NaS1 is upstream of its C terminus, whereas the two interacting proteins, NHERF-1 and NHERF-2, bind at a location closer to the N terminus of NaS1. Moreover NHERF-1 and NHERF-2 increased functional sulfate uptake in Xenopus oocytes when co-expressed with NaS1. Finally we used MYTH 2.0 to demonstrate that the NaPi-IIa transporter homodimerizes via protein-protein interactions within the lipid bilayer. In summary, our study establishes the MYTH 2.0 system as a novel tool for interactive proteomics studies of membrane protein complexes. PMID:18407958

  18. Three ways in, one way out: water dynamics in the trans-membrane domains of the inner membrane translocase AcrB.

    PubMed

    Fischer, Nadine; Kandt, Christian

    2011-10-01

    Powered by proton-motive force, the inner membrane translocase AcrB is the engine of the AcrAB-TolC efflux pump in Escherichia coli. As proton conduction in proteins occurs along hydrogen-bonded networks of polar residues and water molecules, knowledge of the protein-internal water distribution and water-interacting residues allows drawing conclusions to possible pathways of proton conduction. Here, we report a series of 6× 50 ns independent molecular dynamics simulations of asymmetric AcrB embedded in a phospholipid/water environment. Simulating each monomer in its proposed protonation state, we calculated for each trans-membrane domain the average water distribution, identified residues interacting with these waters and quantified each residue's frequency of water hydrogen bond contact. Combining this information we find three possible routes of proton transfer connecting a continuously hydrated region of known key residues in the TMD interior to bulk water by one cytoplasmic and up to three periplasm water channels in monomer B and A. We find that water access of the trans-membrane domains is regulated by four groups of residues in a combination of side chain re-orientations and shifts of trans-membrane helices. Our findings support a proton release event via Arg971 during the C intermediate or in the transition to A, and proton uptake occurring in the A or B state or during a so far unknown intermediate in between B and C where cytoplasmic water access is still possible. Our simulations suggest experimentally testable hypotheses, which have not been investigated so far. PMID:21905112

  19. integrating Solid State NMR and Computations in Membrane Protein Science

    NASA Astrophysics Data System (ADS)

    Cross, Timothy

    2015-03-01

    Helical membrane protein structures are influenced by their native environment. Therefore the characterization of their structure in an environment that models as closely as possible their native environment is critical for achieving not only structural but functional understanding of these proteins. Solid state NMR spectroscopy in liquid crystalline lipid bilayers provides an excellent tool for such characterizations. Two classes of restraints can be obtained - absolute restraints that constrain the structure to a laboratory frame of reference when using uniformly oriented samples (approximately 1° of mosaic spread) and relative restraints that restrain one part of the structure with respect to another part such as torsional and distance restraints. Here, I will discuss unique restraints derived from uniformly oriented samples and the characterization of initial structures utilizing both restraint types, followed by restrained molecular dynamics refinement in the same lipid bilayer environment as that used for the experimental restraint collection. Protein examples will be taken from Influenza virus and Mycobacterium tuberculosis. When available comparisons of structures to those obtained using different membrane mimetic environments will be shown and the causes for structural distortions explained based on an understanding of membrane biophysics and its sophisticated influence on membrane proteins.

  20. Targeting of Tumor Necrosis Factor Receptor 1 to Low Density Plasma Membrane Domains in Human Endothelial Cells*

    PubMed Central

    D'Alessio, Alessio; Kluger, Martin S.; Li, Jie H.; Al-Lamki, Rafia; Bradley, John R.; Pober, Jordan S.

    2010-01-01

    TNFR1 (tumor necrosis factor receptor 1) localizes to caveolae of human endothelial-derived EA.hy926 cells. Transduced TNFR1 molecules lacking amino acid residues 229–244 (spanning the transmembrane/intercellular boundary) are expressed on the cell surface equivalently to full-length TNFR1 molecules but incompletely localize to caveolae. A peptide containing this sequence pulls down CAV-1 (caveolin-1) and TNFR1 from cell lysates but fails to do so following disruption of caveolae with methyl-β-cyclodextrin. We previously reported that methyl-β-cyclodextrin eliminates caveolae and blocks tumor necrosis factor (TNF)-induced internalization of TNFR1 but not TNF-induced activation of NF-κB in EA.hy926 cells. Both CAV-1 and FLOT-2 (flotillin-2), organizing proteins of caveolae and lipid rafts, respectively, associate with caveolae in EA.hy926 cells. Small interfering RNA-mediated knockdown of CAV-1 but not FLOT-2 strikingly reduces caveolae number. Both knockdowns reduce total TNFR1 protein expression, but neither prevents TNFR1 localization to low density membrane domains, TNF-induced internalization of TNFR1, or NF-κB activation by TNF. Both CAV-1 and FLOT-2 knockdowns reduce TNF-mediated activation of stress-activated protein kinase (SAPK). However, both knockdowns reduce expression of TRAF2 (TNF receptor-associated factor-2) protein, and small interfering RNA targeting of TRAF2 also selectively inhibits SAPK activation. We conclude that TNFR1 contains a membrane-proximal sequence that targets the receptor to caveolae/lipid rafts. Neither TNFR1 targeting to nor internalization from these low density membrane domains depends upon CAV-1 or FLOT-2. Furthermore, both NF-κB and SAPK activation appear independent of both TNFR1 localization to low density membrane domains and to TNF-induced receptor internalization. PMID:20511226

  1. Two-dimensional time-domain volume integral equations for scattering of inhomogeneous objects

    NASA Astrophysics Data System (ADS)

    Wang, Jianguo; Fan, Ruyu

    2003-08-01

    This paper proposes a time-domain volume integral equation based method for analyzing the transient scattering from a two-dimensional inhomogeneous cylinder by invoking the volume equivalence principle for both the transverse magnetic and electric cases. The cylinder is discretized into triangular cells, and the electric flux is chosen as the unknown. For the transverse magnetic case, the electric flux is defined on the surfaces of the triangles. For the transverse electric case, because of the electric charges induced inside and on the surface of the cylinder, the electric flux is defined on the edges of the triangles, and expanded in space in terms of two-dimensional surface roof-top basis functions. The time-domain volume integral equation is solved by using a marching-on-in-time scheme. Numerical results obtained using this method are in excellent agreement with the data obtained using the finite-difference time-domain method.

  2. Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

    PubMed Central

    Roche-Håkansson, Hazeline; Håkansson, Anders P.

    2014-01-01

    Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species

  3. Fouling and long-term durability of an integrated forward osmosis and membrane distillation system.

    PubMed

    Husnain, T; Mi, B; Riffat, R

    2015-01-01

    An integrated forward osmosis (FO) and membrane distillation (MD) system has great potential for sustainable wastewater reuse. However, the fouling and long-term durability of the system remains largely unknown. This study investigates the fouling behaviour and efficiency of cleaning procedures of FO and MD membranes used for treating domestic wastewater. Results showed that a significant decline in flux of both FO and MD membranes were observed during treatment of wastewater with organic foulants. However, shear force generated by the increased cross-flow physically removed the loosely attached foulants from the FO membrane surface and resulted in 86-88% recovery of flux by cleaning with tap water. For the MD membrane, almost no flux recovery was achieved due to adsorption of organic foulants on the hydrophobic membrane surface, thus indicating significant irreversible fouling/wetting, which may not be effectively cleaned even with chemical reagents. Long-term (10 d) tests showed consistent performance of the FO membrane by rejecting the contaminants. However, organic foulants reduced the hydrophobicity of the MD membrane, caused wetting problems and allowed contaminants to pass through. The results demonstrate that combination of the FO and MD processes can effectively reduce irreversible membrane fouling and solve the wetting problem of the MD membrane. PMID:26606094

  4. Integrating Moral and Social Development within Middle School Social Studies: A Social Cognitive Domain Approach

    ERIC Educational Resources Information Center

    Nucci, Larry; Creane, Michael W.; Powers, Deborah W.

    2015-01-01

    Eleven teachers and 254 urban middle-school students comprised the sample of this study examining the social and moral development outcomes of the integration of social cognitive domain theory within regular classroom instruction. Participating teachers were trained to construct and implement history lessons that stimulated students' moral…

  5. Integrating Different Perspectives on Socialization Theory and Research: A Domain-Specific Approach

    ERIC Educational Resources Information Center

    Grusec, Joan E.; Davidov, Maayan

    2010-01-01

    There are several different theoretical and research approaches to the study of socialization, characterized by frequently competing basic tenets and apparently contradictory evidence. As a way of integrating approaches and understanding discrepancies, it is proposed that socialization processes be viewed from a domain perspective, with each…

  6. Successful Integration of Membrane Technologies in a Conventional Purification Process of Tannery Wastewater Streams

    PubMed Central

    Stoller, Marco; Sacco, Olga; Sannino, Diana; Chianese, Angelo

    2013-01-01

    The aim of this work is to design and integrate an optimized batch membrane process in a conventional purification process used for the treatment of tannery wastewater. The integration was performed by using two spiral wound membrane modules in series, that is, nanofiltration and reverse osmosis, as substitutes to the biological reactor. The membrane process was designed in terms of sensible fouling issues reduction, which may be observed on the nanofiltration membrane if no optimization is performed. The entity of the fouling phenomena was estimated by pressure cycling measurements, determining both the critical and the threshold flux on the nanofiltration membrane. The obtained results were used to estimate the need of the overdesign of the membrane plant, as well as to define optimized operating conditions in order to handle fouling issues correctly for a long period of time. Finally, the developed membrane process was compared, from a technical and economic point of view, with the conventional biological process, widely offered as an external service near tannery production sites, and, here, proposed to be substituted by membrane technologies. PMID:24956941

  7. TM7SF1 (GPR137B): a novel lysosome integral membrane protein.

    PubMed

    Gao, Jialin; Xia, Libin; Lu, Meiqing; Zhang, Binhua; Chen, Yueping; Xu, Rang; Wang, Lizhuo

    2012-09-01

    In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane. PMID:22729905

  8. Iron Deprivation Affects Drug Susceptibilities of Mycobacteria Targeting Membrane Integrity

    PubMed Central

    Pal, Rahul; Hameed, Saif; Fatima, Zeeshan

    2015-01-01

    Multidrug resistance (MDR) acquired by Mycobacterium tuberculosis (MTB) through continuous deployment of antitubercular drugs warrants immediate search for novel targets and mechanisms. The ability of MTB to sense and become accustomed to changes in the host is essential for survival and confers the basis of infection. A crucial condition that MTB must surmount is iron limitation, during the establishment of infection, since iron is required by both bacteria and humans. This study focuses on how iron deprivation affects drug susceptibilities of known anti-TB drugs in Mycobacterium smegmatis, a “surrogate of MTB.” We showed that iron deprivation leads to enhanced potency of most commonly used first line anti-TB drugs that could be reverted upon iron supplementation. We explored that membrane homeostasis is disrupted upon iron deprivation as revealed by enhanced membrane permeability and hypersensitivity to membrane perturbing agent leading to increased passive diffusion of drug and TEM images showing detectable differences in cell envelope thickness. Furthermore, iron seems to be indispensable to sustain genotoxic stress suggesting its possible role in DNA repair machinery. Taken together, we for the first time established a link between cellular iron and drug susceptibility of mycobacteria suggesting iron as novel determinant to combat MDR. PMID:26779346

  9. Alternative packing of EGFR transmembrane domain suggests that protein-lipid interactions underlie signal conduction across membrane.

    PubMed

    Bocharov, Eduard V; Lesovoy, Dmitry M; Pavlov, Konstantin V; Pustovalova, Yulia E; Bocharova, Olga V; Arseniev, Alexander S

    2016-06-01

    The human epidermal growth factor receptor (EGFR) of HER/ErbB receptor tyrosine kinase family mediates a broad spectrum of cellular responses transducing biochemical signals via lateral dimerization in plasma membrane, while inactive receptors can exist in both monomeric and dimeric forms. Recently, the dimeric conformation of the helical single-span transmembrane domains of HER/ErbB employing the relatively polar N-terminal motifs in a fashion permitting proper kinase activation was experimentally determined. Here we describe the EGFR transmembrane domain dimerization via an alternative weakly polar C-terminal motif A(661)xxxG(665) presumably corresponding to the inactive receptor state. During association, the EGFR transmembrane helices undergo a structural adjustment with adaptation of inter-molecular polar and hydrophobic interactions depending upon the surrounding membrane properties that directly affect the transmembrane helix packing. This might imply that signal transduction through membrane and allosteric regulation are inclusively mediated by coupled protein-protein and protein-lipid interactions, elucidating paradoxically loose linkage between ligand binding and kinase activation. PMID:26903218

  10. Surface Exposure of the HIV-1 Env Cytoplasmic Tail LLP2 Domain during the Membrane Fusion Process

    PubMed Central

    Lu, Lu; Zhu, Yun; Huang, Jinghe; Chen, Xi; Yang, Hengwen; Jiang, Shibo; Chen, Ying-Hua

    2008-01-01

    HIV-1 gp41 cytoplasmic tail (CT) is highly conserved among HIV-1 isolates, particularly the region designated lentivirus lytic peptide (LLP1–2), which includes two α-helical domains LLP1 and LLP2. Although the gp41 CT is recognized as a modulator of viral fusogenicity, little is known about the regulatory mechanism of this region in the viral fusion process. Here we report that anti-LLP1–2 and anti-LLP2 antibodies (IgG) inhibited HIV-1 Env-mediated cell fusion and bound to the interface between effector and target cells at a suboptimal temperature (31.5 °C), which slows down the fusion process and prolongs the fusion intermediate state. This suggests that LLP1–2, especially the LLP2 region located inside the viral membrane, is transiently exposed on the membrane surface during the fusion process. Synthetic LLP2 peptide could bind to the gp41 six-helix bundle core with high binding affinity. These results suggest that the gp41 CT may interact with the gp41 core, via the surface-exposed LLP2 domain, to regulate Env-mediated membrane fusion. PMID:18408000

  11. Autonomous Transmembrane Segment S4 of the Voltage Sensor Domain Partitions into the Lipid Membrane

    PubMed Central

    Tiriveedhi, Venkataswarup; Miller, Melissa; Butko, Peter; Li, Min

    2012-01-01

    The S4 transmembrane segment in voltage-gated ion channels, a highly basic α helix, responds to changes in membrane potential and induces channel opening. Earlier work by others indicates that the S4 segment interacts with lipids in plasma membrane, but its mechanism is unclear. Working with synthetic tryptophan-labeled S4 peptides, we characterized binding of autonomous S4 to lipid membranes. The binding free energy (5.2 ± 0.2 kcal/mol) of the peptide-lipid interaction was estimated from the apparent dissociation constants, determined from the changes in anisotropy of tryptophan fluorescence induced by addition of lipid vesicles with 30 mol% phosphatidylglycerol. The results are in good agreement with the prediction based on the Wimley-White hydrophobicity scale for interfacial (IF) binding of an alpha-helical peptide to the lipid bilayer (6.98 kcal/mol). High salt inhibited the interaction, thus indicating that the peptide/membrane interaction has both electrostatic and non-electrostatic components. Furthermore, the synthetic S4 corresponding to the Shaker potassium channel was found to spontaneously penetrate into the negatively charged lipid membrane to a depth of about 9 Å. Our results revealed important biophysical parameters that influence the interaction of S4 with the membrane: they include fluidity, surface charge, and surface pressure of the membrane, and the α helicity and regular spacing of basic amino-acid residues in the S4 sequence. PMID:22465069

  12. Beyond membrane integrity: Assessing the functionality of human umbilical vein endothelial cells after cryopreservation.

    PubMed

    Marquez-Curtis, Leah A; Sultani, A Billal; McGann, Locksley E; Elliott, Janet A W

    2016-06-01

    Assessment of cell membrane integrity is one of the most widely used methods to measure post-cryopreservation viability of cells such as human umbilical vein endothelial cells (HUVECs). However, an evaluation of cell function provides a better measure of cell quality following cryopreservation. The tube formation assay mimics angiogenesis in vitro and can be used to quantitate the ability of endothelial cells to form capillary-like tubular structures when cultured on reconstituted basement membrane (Matrigel). We compared the membrane integrity (measured by flow cytometry) and tube forming ability of HUVEC suspensions exposed to 10% dimethyl sulfoxide (Me2SO), cooled at 1 °C/min to various sub-zero temperatures, plunged directly into liquid nitrogen, stored for an hour, and thawed rapidly. We found that as membrane integrity increased so did the various parameters associated with the extent of in vitro angiogenesis; however, in comparison to fresh cells with a similar percentage of membrane-intact cells, the extent of tube formation, expressed as total tube length, is significantly lower in previously frozen cells for the lower range of post-thaw membrane integrities. Our findings underscore the value of an assay that quantifies a specific function that a cell is known to perform in vivo to measure the success of cryopreservation protocols. PMID:27182035

  13. On scattered waves and lipid domains: detecting membrane rafts with X-rays and neutrons.

    PubMed

    Marquardt, Drew; Heberle, Frederick A; Nickels, Jonathan D; Pabst, Georg; Katsaras, John

    2015-12-21

    In order to understand the biological role of lipids in cell membranes, it is necessary to determine the mesoscopic structure of well-defined model membrane systems. Neutron and X-ray scattering are non-invasive, probe-free techniques that have been used extensively in such systems to probe length scales ranging from angstroms to microns, and dynamics occurring over picosecond to millisecond time scales. Recent developments in the area of phase separated lipid systems mimicking membrane rafts will be presented, and the underlying concepts of the different scattering techniques used to study them will be discussed in detail. PMID:26428538

  14. On scattered waves and lipid domains: detecting membrane rafts with X-rays and neutrons

    PubMed Central

    Marquardt, Drew; Heberle, Frederick A.; Nickels, Jonathan D.

    2015-01-01

    In order to understand the biological role of lipids in cell membranes, it is necessary to determine the mesoscopic structure of well-defined model membrane systems. Neutron and X-ray scattering are non-invasive, probe-free techniques that have been used extensively in such systems to probe length scales ranging from angstroms to microns, and dynamics occurring over picosecond to millisecond time scales. Recent developments in the area of phase separated lipid systems mimicking membrane rafts will be presented, and the underlying concepts of the different scattering techniques used to study them will be discussed in detail. PMID:26428538

  15. The fission yeast pleckstrin homology domain protein Spo7 is essential for initiation of forespore membrane assembly and spore morphogenesis

    PubMed Central

    Nakamura-Kubo, Michiko; Hirata, Aiko; Shimoda, Chikashi; Nakamura, Taro

    2011-01-01

    Sporulation in fission yeast represents a unique mode of cell division in which a new cell is formed within the cytoplasm of a mother cell. This event is accompanied by formation of the forespore membrane (FSM), which becomes the plasma membrane of spores. At prophase II, the spindle pole body (SPB) forms an outer plaque, from which formation of the FSM is initiated. Several components of the SPB play an indispensable role in SPB modification, and therefore in sporulation. In this paper, we report the identification of a novel SPB component, Spo7, which has a pleckstrin homology (PH) domain. We found that Spo7 was essential for initiation of FSM assembly, but not for SPB modification. Spo7 directly bound to Meu14, a component of the leading edge of the FSM, and was essential for proper localization of Meu14. The PH domain of Spo7 had affinity for phosphatidylinositol 3-phosphate (PI3P). spo7 mutants lacking the PH domain showed aberrant spore morphology, similar to that of meu14 and phosphatidylinositol 3-kinase (pik3) mutants. Our study suggests that Spo7 coordinates formation of the leading edge and initiation of FSM assembly, thereby accomplishing accurate formation of the FSM. PMID:21775631

  16. Integrated Water Gas Shift Membrane Reactors Utilizing Novel, Non Precious Metal Mixed Matrix Membrane

    SciTech Connect

    Ferraris, John

    2013-09-30

    Nanoparticles of zeolitic imidazolate frameworks and other related hybrid materials were prepared by modifying published synthesis procedures by introducing bases, changing stoichiometric ratios, or adjusting reaction conditions. These materials were stable at temperatures >300 °C and were compatible with the polymer matrices used to prepare mixed- matrix membranes (MMMs). MMMs tested at 300 °C exhibited a >30 fold increase in permeability, compared to those measured at 35 °C, while maintaining H{sub 2}/CO{sub 2} selectivity. Measurements at high pressure (up to 30 atm) and high temperature (up to 300 °C) resulted in an increase in gas flux across the membrane with retention of selectivity. No variations in permeability were observed at high pressures at either 35 or 300 °C. CO{sub 2}-induced plasticization was not observed for Matrimid®, VTEC, and PBI polymers or their MMMs at 30 atm and 300 °C. Membrane surface modification by cross-linking with ethanol diamine resulted in an increase in H{sub 2}/CO{sub 2} selectivity at 35 °C. Spectrometric analysis showed that the cross-linking was effective to temperatures <150 °C. At higher temperatures, the cross-linked membranes exhibit a H{sub 2}/CO{sub 2} selectivity similar to the uncross-linked polymer. Performance of the polybenzimidazole (PBI) hollow fibers prepared at Santa Fe Science and Technology (SFST, Inc.) showed increased flux o to a flat PBI membrane. A water-gas shift reactor has been built and currently being optimized for testing under DOE conditions.

  17. Selecting Optimum Eukaryotic Integral Membrane Proteins for Structure Determination by Rapid Expression and Solubilization Screening

    PubMed Central

    Li, Min; Hays, Franklin A.; Roe-Zurz, Zygy; Vuong, Linda; Kelly, Libusha; Ho, Chi-Min; Robbins, Renée M.; Pieper, Ursula; O’Connell, Joseph D.; Miercke, Larry J. W.; Giacomini, Kathleen M.; Sali, Andrej; Stroud, Robert M.

    2009-01-01

    A medium throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from the budding yeast Saccharomyces cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism’s membrane proteome. 272 targets were expressed and of these 234 solubilized in the detergent n-dodecyl-β-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of ten human integral membrane proteins from the solute carrier superfamily (SLC). This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others. PMID:19061901

  18. On the Assessment of Acoustic Scattering and Shielding by Time Domain Boundary Integral Equation Solutions

    NASA Technical Reports Server (NTRS)

    Hu, Fang Q.; Pizzo, Michelle E.; Nark, Douglas M.

    2016-01-01

    Based on the time domain boundary integral equation formulation of the linear convective wave equation, a computational tool dubbed Time Domain Fast Acoustic Scattering Toolkit (TD-FAST) has recently been under development. The time domain approach has a distinct advantage that the solutions at all frequencies are obtained in a single computation. In this paper, the formulation of the integral equation, as well as its stabilization by the Burton-Miller type reformulation, is extended to cases of a constant mean flow in an arbitrary direction. In addition, a "Source Surface" is also introduced in the formulation that can be employed to encapsulate regions of noise sources and to facilitate coupling with CFD simulations. This is particularly useful for applications where the noise sources are not easily described by analytical source terms. Numerical examples are presented to assess the accuracy of the formulation, including a computation of noise shielding by a thin barrier motivated by recent Historical Baseline F31A31 open rotor noise shielding experiments. Furthermore, spatial resolution requirements of the time domain boundary element method are also assessed using point per wavelength metrics. It is found that, using only constant basis functions and high-order quadrature for surface integration, relative errors of less than 2% may be obtained when the surface spatial resolution is 5 points-per-wavelength (PPW) or 25 points-per-wavelength squared (PPW2).

  19. Visualization of Sterol-Rich Membrane Domains with Fluorescently-Labeled Theonellamides

    PubMed Central

    Nishimura, Shinichi; Ishii, Kumiko; Iwamoto, Kunihiko; Arita, Yuko; Matsunaga, Shigeki; Ohno-Iwashita, Yoshiko; Sato, Satoshi B.; Kakeya, Hideaki; Kobayashi, Toshihide; Yoshida, Minoru

    2013-01-01

    Cholesterol plays important roles in biological membranes. The cellular location where cholesterol molecules work is prerequisite information for understanding their dynamic action. Bioimaging probes for cholesterol molecules would be the most powerful means for unraveling the complex nature of lipid membranes. However, only a limited number of chemical or protein probes have been developed so far for cytological analysis. Here we show that fluorescently-labeled derivatives of theonellamides act as new sterol probes in mammalian cultured cells. The fluorescent probes recognized cholesterol molecules and bound to liposomes in a cholesterol-concentration dependent manner. The probes showed patchy distribution in the plasma membrane, while they stained specific organelle in the cytoplasm. These data suggest that fTNMs will be valuable sterol probes for studies on the role of sterols in the biological membrane under a variety of experimental conditions. PMID:24386262

  20. A Novel and Facile Method to Prepare Integrated Electrospun Nanofibrous Membrane with Soldered Junctions.

    PubMed

    Shen, Lingdi; Chen, Jiajia; Hong, Guishan; Wang, Xuefen

    2016-01-01

    Integrated electrospun nanofibrous membrane was prepared by creating soldered junctions between nanofibers via a facile strategy. Polyacrylonitrile (PAN) mixed with poly(vinylidene fluoride) (PVDF) at different ratios of PVDF were prepared in N,N'-dimethyl formamide (DMF), then electrospun to fabricate PAN/PVDF membranes. PVDF can form microgels in DMF which slows down volatile speed of DMF and affects the solidification of PAN/PVDF nanofibers. The resulting membranes were investigated by Fourier transform infrared spectroscopy, scanning electron microscopy, dynamic water contact angle and tensile testing to confirm the morphology and mechanical properties. Soldered junctions were observed between nanofibers with the increase of PVDF content. These junctions made the membrane integrated and greatly enhanced tensile strength from 5.1 to 8.1 MPa (increased by ~60%) and tensile modulus from 49.4 to 117.9 MPa (increased by ~139%) without compromising porosity when the content of PVDF increased from 0 to 60 wt%. PMID:27398532

  1. An equivalent domain integral method for three-dimensional mixed-mode fracture problems

    NASA Technical Reports Server (NTRS)

    Shivakumar, K. N.; Raju, I. S.

    1992-01-01

    A general formulation of the equivalent domain integral (EDI) method for mixed mode fracture problems in cracked solids is presented. The method is discussed in the context of a 3-D finite element analysis. The J integral consists of two parts: the volume integral of the crack front potential over a torus enclosing the crack front and the crack surface integral due to the crack front potential plus the crack face loading. In mixed mode crack problems the total J integral is split into J sub I, J sub II, and J sub III representing the severity of the crack front in three modes of deformations. The direct and decomposition methods are used to separate the modes. These two methods were applied to several mixed mode fracture problems, were analyzed, and results were found to agree well with those available in the literature. The method lends itself to be used as a post-processing subroutine in a general purpose finite element program.

  2. An equivalent domain integral method for three-dimensional mixed-mode fracture problems

    NASA Technical Reports Server (NTRS)

    Shivakumar, K. N.; Raju, I. S.

    1991-01-01

    A general formulation of the equivalent domain integral (EDI) method for mixed mode fracture problems in cracked solids is presented. The method is discussed in the context of a 3-D finite element analysis. The J integral consists of two parts: the volume integral of the crack front potential over a torus enclosing the crack front and the crack surface integral due to the crack front potential plus the crack face loading. In mixed mode crack problems the total J integral is split into J sub I, J sub II, and J sub III representing the severity of the crack front in three modes of deformations. The direct and decomposition methods are used to separate the modes. These two methods were applied to several mixed mode fracture problems, were analyzed, and results were found to agree well with those available in the literature. The method lends itself to be used as a post-processing subroutine in a general purpose finite element program.

  3. Membrane association of the CD3ε signaling domain is required for optimal T cell development and function1

    PubMed Central

    Bettini, Matthew L.; Guy, Clifford; Dash, Pradyot; Vignali, Kate M.; Hamm, David E.; Dobbins, Jessica; Gagnon, Etienne; Thomas, Paul G.; Wucherpfennig, Kai W.; Vignali, Dario A.A.

    2014-01-01

    The T cell receptor (TCR):CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). Here we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4−CD8− double negative-3 (DN3) to DN4 thymocyte transition, due to enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function. PMID:24899501

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

    PubMed

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

    2016-01-01

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

  5. Recombinant expression, purification, and biophysical characterization of the transmembrane and membrane proximal domains of HIV-1 gp41

    PubMed Central

    Gong, Zhen; Kessans, Sarah A; Song, Lusheng; Dörner, Katerina; Lee, Ho-Hsien; Meador, Lydia R; LaBaer, Joshua; Hogue, Brenda G; Mor, Tsafrir S; Fromme, Petra

    2014-01-01

    The transmembrane subunit (gp41) of the envelope glycoprotein of HIV-1 associates noncovalently with the surface subunit (gp120) and together they play essential roles in viral mucosal transmission and infection of target cells. The membrane proximal region (MPR) of gp41 is highly conserved and contains epitopes of broadly neutralizing antibodies. The transmembrane (TM) domain of gp41 not only anchors the envelope glycoprotein complex in the viral membrane but also dynamically affects the interactions of the MPR with the membrane. While high-resolution X-ray structures of some segments of the MPR were solved in the past, they represent the post-fusion forms. Structural information on the TM domain of gp41 is scant and at low resolution. Here we describe the design, expression and purification of a protein construct that includes MPR and the transmembrane domain of gp41 (MPR-TMTEV-6His), which reacts with the broadly neutralizing antibodies 2F5 and 4E10 and thereby may represent an immunologically relevant conformation mimicking a prehairpin intermediate of gp41. The expression level of MPR-TMTEV-6His was improved by fusion to the C-terminus of Mistic protein, yielding ∼1 mg of pure protein per liter. The isolated MPR-TMTEV-6His protein was biophysically characterized and is a monodisperse candidate for crystallization. This work will enable further investigation into the structure of MPR-TMTEV-6His, which will be important for the structure-based design of a mucosal vaccine against HIV-1. PMID:25155369

  6. Cell Membrane Integrity in Myotonic Dystrophy Type 1: Implications for Therapy

    PubMed Central

    González-Barriga, Anchel; Kranzen, Julia; Croes, Huib J. E.; Bijl, Suzanne; van den Broek, Walther J. A. A.; van Kessel, Ingeborg D. G.; van Engelen, Baziel G. M.; van Deutekom, Judith C. T.; Wieringa, Bé; Mulders, Susan A. M.; Wansink, Derick G.

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy. PMID:25799359

  7. Identification of novel families and classification of the C2 domain superfamily elucidate the origin and evolution of membrane targeting activities in eukaryotes.

    PubMed

    Zhang, Dapeng; Aravind, L

    2010-12-01

    Eukaryotes contain an elaborate membrane system, which bounds the cell itself, nuclei, organelles and transient intracellular structures, such as vesicles. The emergence of this system was marked by an expansion of a number of structurally distinct classes of lipid-binding domains that could throw light on the early evolution of eukaryotic membranes. The C2 domain is a useful model to understand these events because it is one of the most prevalent eukaryotic lipid-binding domains deployed in diverse functional contexts. Most studies have concentrated on C2 domains prototyped by those in protein kinase C (PKC-C2) isoforms that bind lipid in a calcium-dependent manner. While two other distinct families of C2 domains, namely those in PI3K-C2 and PTEN-C2 are also recognized, a complete picture of evolutionary relationships within the C2 domain superfamily is lacking. We systematically studied this superfamily using sequence profile searches, phylogenetic and phyletic-pattern analysis and structure-prediction. Consequently, we identified several distinct families of C2 domains including those respectively typified by C2 domains in the Aida (axin interactor, dorsalization associated) proteins, B9 proteins (e.g. Mks1 (Xbx-7), Stumpy (Tza-1) and Tza-2) involved in centrosome migration and ciliogenesis, Dock180/Zizimin proteins which are Rac/CDC42 GDP exchange factors, the EEIG1/Sym-3, EHBP1 and plant RPG/PMI1 proteins involved in endocytotic recycling and organellar positioning and an apicomplexan family. We present evidence that the last eukaryotic common ancestor (LECA) contained at least 10 C2 domains belonging to 6 well-defined families. Further, we suggest that this pre-LECA diversification was linked to the emergence of several quintessentially eukaryotic structures, such as membrane repair and vesicular trafficking system, anchoring of the actin and tubulin cytoskeleton to the plasma and vesicular membranes, localization of small GTPases to membranes and lipid

  8. Identification of Novel Families and Classification of the C2 domain Superfamily Elucidate the Origin and Evolution of Membrane Targeting Activities in Eukaryotes

    PubMed Central

    Zhang, Dapeng; Aravind, L.

    2010-01-01

    Eukaryotes contain an elaborate membrane system, which bounds the cell itself, nuclei, organelles and transient intracellular structures, such as vesicles. The emergence of this system was marked by an expansion of number of structurally distinct classes of lipid-binding domains that could throw light on the early evolution of eukaryotic membranes. The C2 domain is a useful model to understand these events because it is one of the most prevalent eukaryotic lipid-binding domains deployed in diverse functional contexts. Most studies have concentrated on C2 domains prototyped by those in protein kinase C (PKC-C2) isoforms that bind lipid in a calcium-dependent manner. While two other distinct families of C2 domains, namely those in PI3K-C2 and PTEN-C2 are also recognized, a complete picture of evolutionary relationships within the C2 domain superfamily is lacking. We systematically studied this superfamily using sequence-profile searches, phylogenetic and phyletic-pattern analysis and structure-prediction. Consequently, we identified several distinct families of C2 domains including those respectively typified by C2 domains in the Aida (axin interactor, dorsalization associated) proteins, B9 proteins (e.g. Mks1 (Xbx-7), Stumpy (Tza-1) and Tza-2) involved in centrosome migration and ciliogenesis, Dock180/Zizimin proteins which are Rac/CDC42 GDP exchange factors, the EEIG1/Sym-3, EHBP1 and plant RPG/PMI1 proteins involved in endocytotic recycling and organellar positioning and an apicomplexan family. We present evidence that the last eukaryotic common ancestor (LECA) contained at least 10 C2 domains belonging to 6 well-defined families. Further, we suggest that this pre-LECA diversification was linked to emergence of several quintessentially eukaryotic structures, such as membrane repair and vesicular trafficking system, anchoring of the actin and tubulin cytoskeleton to the plasma and vesicular membranes, localization of small GTPases to membranes and lipid

  9. In vitro nanobody discovery for integral membrane protein targets

    PubMed Central

    Doshi, Rupak; Chen, Beverly R.; Vibat, Cecile Rose T.; Huang, Norman; Lee, Chang-Wook; Chang, Geoffrey

    2014-01-01

    Nanobodies (Nbs) or single-domain antibodies are among the smallest and most stable binder scaffolds known. In vitro display is a powerful antibody discovery technique used worldwide. We describe the first adaptation of in vitro mRNA/cDNA display for the rapid, automatable discovery of Nbs against desired targets, and use it to discover the first ever reported nanobody against the human full-length glucose transporter, GLUT-1. We envision our streamlined method as a bench-top platform technology, in combination with various molecular evolution techniques, for expedited Nb discovery. PMID:25342225

  10. Reduced Graphene Oxide Bipolar Membranes for Integrated Solar Water Splitting in Optimal pH.

    PubMed

    McDonald, Michael B; Bruce, Jared P; McEleney, Kevin; Freund, Michael S

    2015-08-24

    The integration of light absorbers and catalysts for the water splitting process requires a membrane capable of both ion and electron management and product separation to realize efficient solar fuels systems. Bipolar membranes can maintain a pH gradient for optimal reaction conditions by the dissociation of water. Such membranes that contain graphene in the interfacial layer are fabricated by the chemical reduction of a uniformly deposited graphene oxide layer to convert sp(3) catalyst regions to sp(2) conductive regions. The resulting electrical and water dissociation properties are optimized by adjusting the exposure conditions, and treatments of less than 5 min render an interface that exceeds the conductivity requirements for integrated solar water splitting and increases the overpotential by <0.3 V. Integration with photoelectrodes is examined by characterizing the electrical interface formed between graphene and Si microwires, and we found that efficient Ohmic junctions are possible. PMID:26204850

  11. An Essential Tyrosine Phosphatase Homolog Regulates Cell Separation, Outer Membrane Integrity, and Morphology in Caulobacter crescentus ▿ †

    PubMed Central

    Shapland, Elaine B.; Reisinger, Sarah J.; Bajwa, Amrita K.; Ryan, Kathleen R.

    2011-01-01

    Although reversible phosphorylation on tyrosine residues regulates the activity of many eukaryotic proteins, there are few examples of this type of regulation in bacteria. We have identified the first essential tyrosine phosphatase homolog in a bacterium, Caulobacter crescentusCtpA. ctpAmutants with altered active-site residues are nonviable, and depletion of CtpA yields chains of cells with blebbed outer membranes, linked by unresolved peptidoglycan. CtpA overexpression reduces cell curvature in a manner similar to deleting the intermediate filament protein crescentin, but it does not disrupt crescentin localization or membrane attachment. Although it has no obvious signal sequence or transmembrane-spanning domains, CtpA associates with the Caulobacterinner membrane. Immunolocalization experiments suggest that CtpA accumulates at the division site during the last quarter of the cell cycle. We propose that CtpA dephosphorylates one or more proteins involved in peptidoglycan biosynthesis or remodeling, which in turn affect cell separation, cell envelope integrity, and vibrioid morphology. PMID:21705597

  12. Integrated capture, transport, and magneto-mechanical resonant sensing of superparamagnetic microbeads using magnetic domain walls.

    PubMed

    Rapoport, E; Montana, D; Beach, G S D

    2012-11-01

    An integrated platform for the capture, transport, and detection of individual superparamagnetic microbeads is described for lab-on-a-chip biomedical applications. Magnetic domain walls in magnetic tracks have previously been shown to be capable of capturing and transporting individual beads through a fluid at high speeds. Here it is shown that the strong magnetostatic interaction between a bead and a domain wall leads to a distinct magneto-mechanical resonance that reflects the susceptibility and hydrodynamic size of the trapped bead. Numerical and analytical modeling is used to quantitatively explain this resonance, and the magneto-mechanical resonant response under sinusoidal drive is experimentally characterized both optically and electrically. The observed bead resonance presents a new mechanism for microbead sensing and metrology. The dual functionality of domain walls as both bead carriers and sensors is a promising platform for the development of lab-on-a-bead technologies. PMID:22955796

  13. The Bone Dysplasia Ontology: integrating genotype and phenotype information in the skeletal dysplasia domain

    PubMed Central

    2012-01-01

    Background Skeletal dysplasias are a rare and heterogeneous group of genetic disorders affecting skeletal development. Patients with skeletal dysplasias suffer from many complex medical issues including degenerative joint disease and neurological complications. Because the data and expertise associated with this field is both sparse and disparate, significant benefits will potentially accrue from the availability of an ontology that provides a shared conceptualisation of the domain knowledge and enables data integration, cross-referencing and advanced reasoning across the relevant but distributed data sources. Results We introduce the design considerations and implementation details of the Bone Dysplasia Ontology. We also describe the different components of the ontology, including a comprehensive and formal representation of the skeletal dysplasia domain as well as the related genotypes and phenotypes. We then briefly describe SKELETOME, a community-driven knowledge curation platform that is underpinned by the Bone Dysplasia Ontology. SKELETOME enables domain experts to use, refine and extend and apply the ontology without any prior ontology engineering experience--to advance the body of knowledge in the skeletal dysplasia field. Conclusions The Bone Dysplasia Ontology represents the most comprehensive structured knowledge source for the skeletal dysplasias domain. It provides the means for integrating and annotating clinical and research data, not only at the generic domain knowledge level, but also at the level of individual patient case studies. It enables links between individual cases and publicly available genotype and phenotype resources based on a community-driven curation process that ensures a shared conceptualisation of the domain knowledge and its continuous incremental evolution. PMID:22449239

  14. Widefield microscopy for live imaging of lipid domains and membrane dynamics

    PubMed Central

    Wheeler, Guy; Tyler, Kevin M.

    2011-01-01

    Within the lateral organisation of plasma membranes of polarized cell types there exist heterogenous microdomains of distinct lipid composition, the small size of which (10–200 nm) makes them difficult to discern with traditional microscopic techniques, but which can be distinguished on the basis of lipid packing. These microdomains or rafts can be concentrated in larger more visible liquid-ordered regions, particularly by cross-linking of their constituents as in the immunological synapse or in features of the polarized cell such as pseudopodia or flagella. One technique, Laurdan fluorescence microscopy, has proven very useful for distinguishing such regions but has hitherto relied on 2-photon confocal microscopy. This has to some extent limited its utility to living systems and its widespread adoption in studying membrane dynamics on the surface of living cells. Here we describe and validate the adaptation of a standard widefield fluorescence microscope for live imaging of Laurdan stained cell membranes. PMID:21126508

  15. Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

    PubMed Central

    Lin, Qingqing; London, Erwin

    2013-01-01

    Because transmembrane (TM) protein localization, or nonlocalization, in ordered membrane domains (rafts) is a key to understanding membrane domain function, it is important to define the origin of protein-raft interaction. One hypothesis is that a tight noncovalent attachment of TM proteins to lipids that have a strong affinity for ordered domains can be sufficient to induce raft-protein interaction. The sterol-binding protein perfringolysin O (PFO) was used to test this hypothesis. PFO binds both to sterols that tend to localize in ordered domains (e.g., cholesterol), and to those that do not (e.g., coprostanol), but it does not bind to epicholesterol, a raft-promoting 3α-OH sterol. Using a fluorescence resonance energy transfer assay in model membrane vesicles containing coexisting ordered and disordered lipid domains, both TM and non-TM forms of PFO were found to concentrate in ordered domains in vesicles containing high and low-Tm lipids plus cholesterol or 1:1 (mol/mol) cholesterol/epicholesterol, whereas they concentrate in disordered domains in vesicles containing high-Tm and low-Tm lipids plus 1:1 (mol/mol) coprostanol/epicholesterol. Combined with previous studies this behavior indicates that TM protein association with ordered domains is dependent upon both the association of the protein-bound sterol with ordered domains and hydrophobic match between TM segments and rafts. PMID:24359745

  16. PERIPARTUM DEPRESSION AND ANXIETY AS AN INTEGRATIVE CROSS DOMAIN TARGET FOR PSYCHIATRIC PREVENTATIVE MEASURES

    PubMed Central

    Babb, Jessica A.; Deligiannidis, Kristina M.; Murgatroyd, Christopher A.

    2014-01-01

    Exposure to high levels of early life stress has been identified as a potent risk factor for neurodevelopmental delays in infants, behavioral problems and autism in children, but also for several psychiatric illnesses in adulthood, such as depression, anxiety, autism, and posttraumatic stress disorder. Despite having robust adverse effects on both mother and infant, the pathophysiology of peripartum depression and anxiety are poorly understood. The objective of this review is to highlight the advantages of using an integrated approach addressing several behavioral domains in both animal and clinical studies of peripartum depression and anxiety. It is postulated that a greater focus on integrated cross domain studies will lead to advances in treatments and preventative measures for several disorders associated with peripartum depression and anxiety. PMID:24709228

  17. Attentional selection in visual perception, memory and action: a quest for cross-domain integration

    PubMed Central

    Schneider, Werner X.; Einhäuser, Wolfgang; Horstmann, Gernot

    2013-01-01

    For decades, the cognitive and neural sciences have benefitted greatly from a separation of mind and brain into distinct functional domains. The tremendous success of this approach notwithstanding, it is self-evident that such a view is incomplete. Goal-directed behaviour of an organism requires the joint functioning of perception, memory and sensorimotor control. A prime candidate for achieving integration across these functional domains are attentional processes. Consequently, this Theme Issue brings together studies of attentional selection from many fields, both experimental and theoretical, that are united in their quest to find overreaching integrative principles of attention between perception, memory and action. In all domains, attention is understood as combination of competition and priority control (‘bias’), with the task as a decisive driving factor to ensure coherent goal-directed behaviour and cognition. Using vision as the predominant model system for attentional selection, many studies of this Theme Issue focus special emphasis on eye movements as a selection process that is both a fundamental action and serves a key function in perception. The Theme Issue spans a wide range of methods, from measuring human behaviour in the real word to recordings of single neurons in the non-human primate brain. We firmly believe that combining such a breadth in approaches is necessary not only for attentional selection, but also to take the next decisive step in all of the cognitive and neural sciences: to understand cognition and behaviour beyond isolated domains. PMID:24018715

  18. Membrane binding mode of intrinsically disordered cytoplasmic domains of T cell receptor signaling subunits depends on lipid composition

    SciTech Connect

    Sigalov, Alexander B.; Hendricks, Gregory M.

    2009-11-13

    Intrinsically disordered cytoplasmic domains of T cell receptor (TCR) signaling subunits including {zeta}{sub cyt} and CD3{epsilon}{sub cyt} all contain one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor triggering. Membrane binding-induced helical folding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} ITAMs is thought to control TCR activation. However, the question whether or not lipid binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} is necessarily accompanied by a folding transition of ITAMs remains open. In this study, we investigate whether the membrane binding mechanisms of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depend on the membrane model used. Circular dichroic and fluorescence data indicate that binding of {zeta}{sub cyt} and CD3{epsilon}{sub cyt} to detergent micelles and unstable vesicles is accompanied by a disorder-to-order transition, whereas upon binding to stable vesicles these proteins remain unfolded. Using electron microscopy and dynamic light scattering, we show that upon protein binding, unstable vesicles fuse and rupture. In contrast, stable vesicles remain intact under these conditions. This suggests different membrane binding modes for {zeta}{sub cyt} and CD3{epsilon}{sub cyt} depending on the bilayer stability: (1) coupled binding and folding, and (2) binding without folding. These findings explain the long-standing puzzle in the literature and highlight the importance of the choice of an appropriate membrane model for protein-lipid interactions studies.

  19. The use of time-domain integral equations in electromagnetism problems

    NASA Astrophysics Data System (ADS)

    Berthon, A.; Vallet, E.

    1984-10-01

    The application of time-domain integral equations to the analysis of the scattering and propagation of EM fields is surveyed. Explicit iterative time-stepping procedures are developed for such problems as simple-conductor obstacles, one-dimensional obstacles, orifices, small obstacles, and media of finite conductivity (interface fields, reflection factors, and shielding problems). Numerical results for problems involving the interaction of a dipole field with various cylindrical obstacles are presented in graphs and diagrams.

  20. Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family.

    PubMed

    Shanklin, John; Whittle, Edward

    2003-06-19

    Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site. PMID:12804773

  1. The interaction between the pleckstrin homology domain of ceramide kinase and phosphatidylinositol 4,5-bisphosphate regulates the plasma membrane targeting and ceramide 1-phosphate levels

    SciTech Connect

    Kim, Tack-Joong; Mitsutake, Susumu; Igarashi, Yasuyuki . E-mail: yigarash@pharm.hokudai.ac.jp

    2006-04-07

    Ceramide kinase (CERK) converts ceramide to ceramide-1-phosphate (C1P), which has recently emerged as a new bioactive molecule capable of regulating diverse cellular functions. The N-terminus of the CERK protein encompasses a sequence motif known as a pleckstrin homology (PH) domain. Although the PH domain was previously demonstrated to be an important domain for the subcellular localization of CERK, the precise properties of this domain remained unclear. In this study, we reveal that the PH domain of CERK exhibits high affinity for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P{sub 2}), among other lipids. Furthermore, in COS7 cells, GFP-fused CERK translocated rapidly from the cytoplasm to the plasma membrane in response to hyper-osmotic stress, which is known to increase the intracellular PI(4,5)P{sub 2} levels, whereas a PH domain deletion mutant did not. Additionally, in [{sup 32}P]orthophosphate-labeled COS7 cells, the translocation of CERK to the plasma membrane induced a 2.8-fold increase in C1P levels. The study presented here provides insight into the crucial role of the CERK-PH domain in plasma membrane targeting, through its binding to PI(4,5)P{sub 2}, and subsequent induction of C1P production in the vicinity of the membrane.

  2. Binding of basic peptides to membranes produces lateral domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol 4,5-bisphosphate: an electrostatic model and experimental results.

    PubMed Central

    Denisov, G; Wanaski, S; Luan, P; Glaser, M; McLaughlin, S

    1998-01-01

    Direct fluorescence digital imaging microscopy observations demonstrate that a basic peptide corresponding to the effector region of the myristoylated alanine-rich C kinase substrate (MARCKS) self-assembles into membrane domains enriched in the acidic phospholipids phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2). We show here that pentalysine, which corresponds to the first five residues of the MARCKS effector region peptide and binds to membranes through electrostatic interactions, also forms domains enriched in PS and PIP2. We present a simple model of domain formation that represents the decrease in the free energy of the system as the sum of two contributions: the free energy of mixing of neutral and acidic lipids and the electrostatic free energy. The first contribution is always positive and opposes domain formation, whereas the second contribution may become negative and, at low ionic strength, overcome the first contribution. Our model, based on Gouy-Chapman-Stern theory, makes four predictions: 1) multivalent basic ligands, for which the membrane binding is a steep function of the mole fraction of acidic lipid, form domains enriched in acidic lipids; domains break up at high concentrations of either 2) basic ligand or 3) monovalent salt; and 4) if multivalent anionic lipids (e.g., PIP2) are present in trace concentrations in the membrane, they partition strongly into the domains. These predictions agree qualitatively with experimental data obtained with pentalysine and spermine, another basic ligand. PMID:9533686

  3. Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

    PubMed Central

    Valentine, Cathleen D.; Haggie, Peter M.

    2011-01-01

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes. PMID:21680711

  4. Development and application of a procedure for evaluating the long-term integrity of membranes for the anaerobic fluidized membrane bioreactor (AFMBR).

    PubMed

    Shin, Chungheon; Kim, Kihyun; McCarty, Perry L; Kim, Jeonghwan; Bae, Jaeho

    2016-01-01

    A bench-scale short-term test, developed to predict the long-term integrity of membranes with potential for use in anaerobic fluidized-bed membrane bioreactors, was used to evaluate several commercial hollow-fiber membranes. It was found that membrane performance varied widely, some membranes failing much more rapidly than others. Also found was that larger sizes of the fluidized media, in this case granular activated carbon (GAC), severely affected membrane structural integrity more than did smaller sizes, as did the method used for membrane attachment. Within the limits studied, the GAC packing ratio had only a minor impact. A decrease in membrane permeability that sometimes resulted during the testing and was caused by the deposition of fine GAC particles could be eliminated without membrane damage through simultaneous chemical cleaning and sonication. This new testing procedure should be useful for selecting membranes and reactor operating conditions to better ensure long-term operating performance of anaerobic fluidized-bed membrane bioreactors. PMID:27438251

  5. Intrasurgical Human Retinal Imaging With Manual Instrument Tracking Using a Microscope-Integrated Spectral-Domain Optical Coherence Tomography Device

    PubMed Central

    Hahn, Paul; Carrasco-Zevallos, Oscar; Cunefare, David; Migacz, Justin; Farsiu, Sina; Izatt, Joseph A.; Toth, Cynthia A.

    2015-01-01

    Purpose: To characterize the first in-human intraoperative imaging using a custom prototype spectral-domain microscope-integrated optical coherence tomography (MIOCT) device during vitreoretinal surgery with instruments in the eye. Methods: Under institutional review board approval for a prospective intraoperative study, MIOCT images were obtained at surgical pauses with instruments held static in the vitreous cavity and then concurrently with surgical maneuvers. Postoperatively, MIOCT images obtained at surgical pauses were compared with images obtained with a high-resolution handheld spectral-domain OCT (HHOCT) system with objective endpoints, including acquisition of images acceptable for analysis and identification of predefined macular morphologic or pathologic features. Results: Human MIOCT images were successfully obtained before incision and during pauses in surgical maneuvers. MIOCT imaging confirmed preoperative diagnoses, such as epiretinal membrane, full-thickness macular hole, and vitreomacular traction and demonstrated successful achievement of surgical goals. MIOCT and HHOCT images obtained at surgical pauses in two cohorts of five patients were comparable with greater than or equal to 80% correlation in 80% of patients. Real-time video-imaging concurrent with surgical manipulations enabled, for the first time using this device, visualization of dynamic instrument-retina interaction with targeted OCT tracking. Conclusion: MIOCT is successful for imaging at surgical pauses and for real-time image guidance with implementation of targeted OCT tracking. Even faster acquisition speeds are currently being developed with incorporation of a swept-source MIOCT engine. Further refinements and investigations will be directed toward continued integration for real-time volumetric imaging of surgical maneuvers. Translational Relevance: Ongoing development of seamless MIOCT systems will likely transform surgical visualization, approaches, and decision-making. PMID

  6. A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues

    PubMed Central

    Mann, Thomas H.; Seth Childers, W.; Blair, Jimmy A.; Eckart, Michael R.; Shapiro, Lucy

    2016-01-01

    All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-type-specific gene regulation. PMID:27117914

  7. A cell cycle kinase with tandem sensory PAS domains integrates cell fate cues.

    PubMed

    Mann, Thomas H; Seth Childers, W; Blair, Jimmy A; Eckart, Michael R; Shapiro, Lucy

    2016-01-01

    All cells must integrate sensory information to coordinate developmental events in space and time. The bacterium Caulobacter crescentus uses two-component phospho-signalling to regulate spatially distinct cell cycle events through the master regulator CtrA. Here, we report that CckA, the histidine kinase upstream of CtrA, employs a tandem-PAS domain sensor to integrate two distinct spatiotemporal signals. Using CckA reconstituted on liposomes, we show that one PAS domain modulates kinase activity in a CckA density-dependent manner, mimicking the stimulation of CckA kinase activity that occurs on its transition from diffuse to densely packed at the cell poles. The second PAS domain interacts with the asymmetrically partitioned second messenger cyclic-di-GMP, inhibiting kinase activity while stimulating phosphatase activity, consistent with the selective inactivation of CtrA in the incipient stalked cell compartment. The integration of these spatially and temporally regulated signalling events within a single signalling receptor enables robust orchestration of cell-type-specific gene regulation. PMID:27117914

  8. Multiscale simulations on conformational dynamics and membrane interactions of the non-structural 2 (NS2) transmembrane domain.

    PubMed

    Hung, Huynh Minh; Hang, Tran Dieu; Nguyen, Minh Tho

    2016-09-01

    Hepatitis C virus (HCV) is one of the most crucial global health issues, in which the HCV non-structural protein 2 (NS2), particularly its three transmembrane segments, plays a crucial role in HCV assembly. In this context, multiscale MD simulations have been applied to investigate the preferred orientation of transmembrane domain of NS2 protein (TNS2) in a POPC bilayer, structural stability and characteristic of intramembrane protein-lipid and protein-protein interaction. Our study indicates that NS2 protein adopts three trans-membrane segments with highly stable α-helix structure in a POPC bilayer and a short helical luminal segment. While the first and second TM segment involved in continuous helical domain, the third TM segment is however cleaved into two sub-segments with different tilt angles via a kink at L87G88. Salt bridges K81-E45, R32-PO4 and R43-PO4 are determined as the key factor to stabilize the structure of TM2 and TM3 which consist of charged residues located in the hydrophobic region of the membrane. PMID:27444387

  9. Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains[S

    PubMed Central

    Bhat, Hema Balakrishna; Kishimoto, Takuma; Abe, Mitsuhiro; Makino, Asami; Inaba, Takehiko; Murate, Motohide; Dohmae, Naoshi; Kurahashi, Atsushi; Nishibori, Kozo; Fujimori, Fumihiro; Greimel, Peter; Ishitsuka, Reiko; Kobayashi, Toshihide

    2013-01-01

    A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes. PMID:23918047

  10. Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia

    NASA Astrophysics Data System (ADS)

    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.

  11. An Effector Domain Mutant of Arf6 Implicates Phospholipase D in Endosomal Membrane RecyclingD⃞

    PubMed Central

    Jovanovic, Olivera A.; Brown, Fraser D.; Donaldson, Julie G.

    2006-01-01

    In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment. PMID:16280360

  12. Master curve captures the effect of domain morphology on ethanol pervaporation through block copolymer membranes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report on the effect of changing nanoscale morphology on pervaporation of ethanol/water mixtures through block copolymer membranes. Experiments were conducted using polystyrene-b-polybutadiene-b-polystyrene (SBS) copolymers with polybutadiene (PB) as the ethanol transporting block, using an 8 wt%...

  13. Comparative sequence analysis of domain I of Plasmodium falciparum apical membrane antigen 1 from Saudi Arabia and worldwide isolates.

    PubMed

    Al-Qahtani, Ahmed A; Abdel-Muhsin, Abdel-Muhsin A; Bin Dajem, Saad M; AlSheikh, Adel Ali H; Bohol, Marie Fe F; Al-Ahdal, Mohammed N; Putaporntip, Chaturong; Jongwutiwes, Somchai

    2016-04-01

    The apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) plays a crucial role in erythrocyte invasion and is a target of protective antibodies. Although domain I of PfAMA1 has been considered a promising vaccine component, extensive sequence diversity in this domain could compromise an effective vaccine design. To explore the extent of sequence diversity in domain I of PfAMA1, P. falciparum-infected blood samples from Saudi Arabia collected between 2007 and 2009 were analyzed and compared with those from worldwide parasite populations. Forty-six haplotypes and a novel codon change (M190V) were found among Saudi Arabian isolates. The haplotype diversity (0.948±0.004) and nucleotide diversity (0.0191±0.0008) were comparable to those from African hyperendemic countries. Positive selection in domain I of PfAMA1 among Saudi Arabian parasite population was observed because nonsynonymous nucleotide substitutions per nonsynonymous site (dN) significantly exceeded synonymous nucleotide substitutions per synonymous site (dS) and Tajima's D and its related statistics significantly deviated from neutrality in the positive direction. Despite a relatively low prevalence of malaria in Saudi Arabia, a minimum of 17 recombination events occurred in domain I. Genetic differentiation was significant between P. falciparum in Saudi Arabia and parasites from other geographic origins. Several shared or closely related haplotypes were found among parasites from different geographic areas, suggesting that vaccine derived from multiple shared epitopes could be effective across endemic countries. PMID:26867816

  14. VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1.

    PubMed

    Ren, Hong Yu; Grove, Diane E; De La Rosa, Oxana; Houck, Scott A; Sopha, Pattarawut; Van Goor, Fredrick; Hoffman, Beth J; Cyr, Douglas M

    2013-10-01

    Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR. PMID:23924900

  15. VX-809 corrects folding defects in cystic fibrosis transmembrane conductance regulator protein through action on membrane-spanning domain 1

    PubMed Central

    Ren, Hong Yu; Grove, Diane E.; De La Rosa, Oxana; Houck, Scott A.; Sopha, Pattarawut; Van Goor, Fredrick; Hoffman, Beth J.; Cyr, Douglas M.

    2013-01-01

    Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR. PMID:23924900

  16. A Key Claudin Extracellular Loop Domain is Critical for Epithelial Barrier Integrity

    PubMed Central

    Mrsny, Randall J.; Brown, G. Thomas; Gerner-Smidt, Kirsten; Buret, Andre G.; Meddings, Jon B.; Quan, Clifford; Koval, Michael; Nusrat, Asma

    2008-01-01

    Intercellular tight junctions (TJs) regulate epithelial barrier properties. Claudins are major structural constituents of TJs and belong to a large family of tetra-spanning membrane proteins that have two predicted extracellular loops (ELs). Given that claudin-1 is widely expressed in epithelia, we further defined the role of its EL domains in determining TJ function. The effects of several claudin-1 EL mimetic peptides on epithelial barrier structure and function were examined. Incubation of model human intestinal epithelial cells with a 27-amino acid peptide corresponding to a portion of the first EL domain (Cldn-153–80) reversibly interfered with epithelial barrier function by inducing the rearrangement of key TJ proteins: occludin, claudin-1, junctional adhesion molecule-A, and zonula occludens-1. Cldn-153–80 associated with both claudin-1 and occludin, suggesting both the direct interference with the ability of these proteins to assemble into functional TJs and their close interaction under physiological conditions. These effects were specific for Cldn-153–80, because peptides corresponding to other claudin-1 EL domains failed to influence TJ function. Furthermore, the oral administration of Cldn-153–80 to rats increased paracellular gastric permeability. Thus, the identification of a critical claudin-1 EL motif, Cldn-153–80, capable of regulating TJ structure and function, offers a useful adjunct to treatments that require drug delivery across an epithelial barrier. PMID:18349130

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

    PubMed Central

    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

  18. Breakdown of interlocking domains may contribute to formation of membranous globules and lens opacity in ephrin-A5(-/-) mice.

    PubMed

    Biswas, Sondip; Son, Alexander; Yu, Qili; Zhou, Renping; Lo, Woo-Kuen

    2016-04-01

    Ephrin-A5, a ligand of the Eph family of receptor tyrosine kinases, plays a key role in lens fiber cell packing and cell-cell adhesion, with approximately 87% of ephrin-A5(-/-) mice develop nuclear cataracts. Here, we investigated the extensive formation of light-scattering globules associated with breakdown of interlocking protrusions during lens opacification in ephrin-A5(-/-) mice. Lenses from wild-type (WT) and ephrin-A5(-/-) mice between 2 and 21 weeks old were studied with light and electron microscopy, immunofluorescence labeling, freeze-fracture TEM and filipin cytochemistry for membrane cholesterol detection. Lens opacities with various densities were first observed in ephrin-A5(-/-) mice at around 60 days old. Dense cataracts in the mutant lenses were seen primarily in the nuclear region surrounded by transparent cortices from all eyes examined. We confirmed that a majority of nuclear cataracts were dislocated posteriorly and ruptured the thinner posterior lens capsule. SEM analysis indicated that numerous interlocking protrusions and wavy ridge-and-valley membrane surfaces in deep cortical and nuclear fibers did not cause lens opacity in both transparent ephrin-A5(-/-) and WT mice. In contrast, abundant isolated membranous globules of approximately 1000 nm in size were distributed randomly along the intact fiber cells during early stage of all ephrin-A5(-/-) cataracts examined. A further examination using both SEM and TEM revealed that isolated globules were generated from the disintegrated interlocking protrusions originally located along the corners of hexagonal fiber cells. Freeze-fracture TEM further revealed the association of square-array aquaporin junctions with both isolated globules and interlocking membrane domains. This study reports for the first time that disrupted interlocking protrusions are the source of numerous large membranous globules that contribute to light scattering and nuclear cataracts in the ephrin-A5(-/-) mice. Our results

  19. The cation channel mucolipin-1 is a bifunctional protein that facilitates membrane remodeling via its serine lipase domain

    PubMed Central

    LaPlante, Janice M.; Falardeau, John L.; Brown, Edward M.; Slaugenhaupt, Susan A.; Vassilev, Peter M.

    2011-01-01

    Phospholipase modulators have been shown to affect the topology of lipid bilayers and the formation of tubulo-vesicular structures, but the specific endogenous phospholipases involved have yet to be identified. Here we show that TRPML1 (MLN1), a Ca2+-permeable channel contributes to membrane remodeling through a serine-lipase consensus domain, and thus represents a novel type of bifunctional protein. Remarkably, this serine lipase active site determines the ability of MLN1 to generate tubulo-vesicular extensions in mucolipin-1-expressing oocytes, human fibroblasts and model membrane vesicles. Our demonstration that MLN1 is involved in membrane remodeling and the formation of extensions suggests that it may play a role in the formation of cellular processes linked to the late endosome/lysosome (LE/L) pathway. MLN1 is absent or mutated in patients with mucolipidosis IV (MLIV), a lysosomal disorder with devastating neurological and other consequences. This study provides potential insight into the pathophysiology of MLIV. PMID:21256127

  20. The Structure and Interactions of Periplasmic Domains of Crucial MmpL Membrane Proteins from Mycobacterium tuberculosis.

    PubMed

    Chim, Nicholas; Torres, Rodrigo; Liu, Yuqi; Capri, Joe; Batot, Gaëlle; Whitelegge, Julian P; Goulding, Celia W

    2015-08-20

    Mycobacterium tuberculosis mycobacterial membrane protein large (MmpL) proteins are important in substrate transport across the inner membrane. Here, we show that MmpL proteins are classified into two phylogenetic clusters, where MmpL cluster II contains three soluble domains (D1, D2, and D3) and has two full-length members, MmpL3 and MmpL11. Significantly, MmpL3 is currently the most druggable M. tuberculosis target. We have solved the 2.4-Å MmpL11-D2 crystal structure, revealing structural homology to periplasmic porter subdomains of RND (multidrug) transporters. The resulting predicted cluster II MmpL membrane topology has D1 and D2 residing, and possibly interacting, within the periplasm. Crosslinking and biolayer interferometry experiments confirm that cluster II D1 and D2 bind with weak affinities, and guided D1-D2 heterodimeric model assemblies. The predicted full-length MmpL3 and MmpL11 structural models reveal key substrate binding and transport residues, and may serve as templates to set the stage for in silico anti-tuberculosis drug development. PMID:26278184

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

    SciTech Connect

    Shang Liang; Hunter, Eric

    2010-09-01

    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.

  2. Role of the transmembrane domain in SNARE protein mediated membrane fusion: peptide nucleic acid/peptide model systems.

    PubMed

    Wehland, Jan-Dirk; Lygina, Antonina S; Kumar, Pawan; Guha, Samit; Hubrich, Barbara E; Jahn, Reinhard; Diederichsen, Ulf

    2016-08-16

    Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by Soluble NSF (N-ethylmaleimide-sensitive factor) Attachment Protein Receptor proteins also known as SNAREs. The backbone of this essential process is the assembly of SNAREs from opposite membranes into tight four helix bundles forcing membranes in close proximity. With model systems resembling SNAREs with reduced complexity we aim to understand how these proteins work at the molecular level. Here, peptide nucleic acids (PNAs) are used as excellent candidates for mimicking the SNARE recognition motif by forming well-characterized duplex structures. Hybridization between complementary PNA strands anchored in liposomes through native transmembrane domains (TMDs) induces the merger of the outer leaflets of the participating vesicles but not of the inner leaflets. A series of PNA/peptide hybrids differing in the length of TMDs and charges at the C-terminal end is presented. Interestingly, mixing of both outer and inner leaflets is seen for TMDs containing an amide in place of the natural carboxylic acid at the C-terminal end. Charged side chains at the C-terminal end of the TMDs are shown to have a negative impact on the mixing of liposomes. The length of the TMDs is vital for fusion as with the use of shortened TMDs, fusion was completely prevented. PMID:27345759

  3. The membrane-spanning domains of caveolins-1 and -2 mediate the formation of caveolin hetero-oligomers. Implications for the assembly of caveolae membranes in vivo.

    PubMed

    Das, K; Lewis, R Y; Scherer, P E; Lisanti, M P

    1999-06-25

    The mammalian caveolin gene family consists of caveolins-1, -2, and -3. The expression of caveolin-3 is muscle-specific. In contrast, caveolins-1 and -2 are co-expressed, and they form a hetero-oligomeric complex in many cell types, with particularly high levels in adipocytes, endothelial cells, and fibroblasts. These caveolin hetero-oligomers are thought to represent the functional assembly units that drive caveolae formation in vivo. Here, we investigate the mechanism by which caveolins-1 and -2 form hetero-oligomers. We reconstituted this reciprocal interaction in vivo and in vitro using a variety of complementary approaches, including the generation of glutathione S-transferase fusion proteins and synthetic peptides. Taken together, our results indicate that the membrane-spanning domains of both caveolins-1 and -2 play a critical role in mediating their ability to interact with each other. This is the first demonstration that these unusual membrane-spanning regions found in the caveolin family play a specific role in protein-protein interactions. PMID:10373486

  4. Integral fill yarn insertion and beatup method using inflatable membrane

    NASA Technical Reports Server (NTRS)

    Farley, Gary L. (Inventor)

    1993-01-01

    An apparatus and method for integral fill yarn insertion and beatup are disclosed. A modified rapier contains a channel for holding fill yarn. The channel is covered with a flexible and inflatable boot, and an inflating apparatus for this boot is also attached. Fill yarn is inserted into the channel, and the rapier is extended into a shed formed by warp yarn. Next, the rapier is pushed into the fell of the fabric, and the flexible and inflatable cover inflated, which both pushes the yarn into the fell of the fabric and performs beatup. The rapier is withdrawn and the shed closed to complete one step of the weaving process.

  5. Microbial responses to membrane cleaning using sodium hypochlorite in membrane bioreactors: Cell integrity, key enzymes and intracellular reactive oxygen species.

    PubMed

    Han, Xiaomeng; Wang, Zhiwei; Wang, Xueye; Zheng, Xiang; Ma, Jinxing; Wu, Zhichao

    2016-01-01

    Sodium hypochlorite (NaClO) is a commonly used reagent for membrane cleaning in membrane bioreactors (MBRs), while it, being a kind of disinfectant (oxidant), may impair viability of microbes or even totally inactivate them upon its diffusion into mixed liquor during membrane cleaning. In this study, we systematically examine the effects of NaClO on microorganisms in terms of microbial cell integrity, metabolism behaviours (key enzymes), and intracellular reactive oxygen species (ROS) under various NaClO concentrations. Different proportions of microbial cells in activated sludge were damaged within several minutes dependent on NaClO dosages (5-50 mg/g-SS), and correspondingly organic matters were released to bulk solution. Inhibition of key enzymes involved in organic matter biodegradation, nitrification and denitrification was observed in the presence of NaClO above 1 mg/g-SS, and thus organic matter and nitrogen removal efficiencies were decreased. It was also demonstrated that intracellular ROS production was increased with the NaClO dosage higher than 1 mg/g-SS, which likely induced further damage to microbial cells. PMID:26512807

  6. Organization of the Escherichia coli aerobic enzyme complexes of oxidative phosphorylation in dynamic domains within the cytoplasmic membrane

    PubMed Central

    Erhardt, Heiko; Dempwolff, Felix; Pfreundschuh, Moritz; Riehle, Marc; Schäfer, Caspar; Pohl, Thomas; Graumann, Peter; Friedrich, Thorsten

    2014-01-01

    The Escherichia coli cytoplasmic membrane contains the enzyme complexes of oxidative phosphorylation (OXPHOS). Not much is known about their supramolecular organization and their dynamics within the membrane in this model organism. In mitochondria and other bacteria, it was demonstrated by nondenaturing electrophoretic methods and electron microscopy that the OXPHOS complexes are organized in so-called supercomplexes, stable assemblies with a defined number of the individual enzyme complexes. To investigate the organization of the E. coli enzyme complexes of aerobic OXPHOS in vivo, we established fluorescent protein fusions of the NADH:ubiquinone oxidoreductase, the succinate:ubiquinone oxidoreductase, the cytochrome bd-I, and the cytochrome bo3 terminal oxidases, and the FoF1 ATP-synthase. The fusions were integrated in the chromosome to prevent artifacts caused by protein overproduction. Biochemical analysis revealed that all modified complexes were fully assembled, active, and stable. The distribution of the OXPHOS complexes in living cells was determined using total internal reflection fluorescence microscopy. The dynamics within the membrane were detected by fluorescence recovery after photobleaching. All aerobic OXPHOS complexes showed an uneven distribution in large mobile patches within the E. coli cytoplasmic membrane. It is discussed whether the individual OXPHOS complexes are organized as clustered individual complexes, here called “segrazones.” PMID:24729508

  7. Prediction of Multi-Type Membrane Proteins in Human by an Integrated Approach

    PubMed Central

    Chen, Lei; Zhang, Ning; Huang, Tao; Cai, Yu-Dong

    2014-01-01

    Membrane proteins were found to be involved in various cellular processes performing various important functions, which are mainly associated to their types. However, it is very time-consuming and expensive for traditional biophysical methods to identify membrane protein types. Although some computational tools predicting membrane protein types have been developed, most of them can only recognize one kind of type. Therefore, they are not as effective as one membrane protein can have several types at the same time. To our knowledge, few methods handling multiple types of membrane proteins were reported. In this study, we proposed an integrated approach to predict multiple types of membrane proteins by employing sequence homology and protein-protein interaction network. As a result, the prediction accuracies reached 87.65%, 81.39% and 70.79%, respectively, by the leave-one-out test on three datasets. It outperformed the nearest neighbor algorithm adopting pseudo amino acid composition. The method is anticipated to be an alternative tool for identifying membrane protein types. New metrics for evaluating performances of methods dealing with multi-label problems were also presented. The program of the method is available upon request. PMID:24676214

  8. Cell-free Expression and In Meso Crystallisation of an Integral Membrane Kinase for Structure Determination

    PubMed Central

    Shah, Syed Tasadaque Ali; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank; Caffrey, Martin

    2014-01-01

    Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a 3-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipidic mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28 Å resolution. The quality of cellular and cell-free expressed kinase samples have been evaluated systematically by comparing i) spectroscopic properties, ii) purity and oligomer formation, iii) lipid content and iv) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved. PMID:25012698

  9. Cell-free expression and in meso crystallisation of an integral membrane kinase for structure determination.

    PubMed

    Boland, Coilín; Li, Dianfan; Shah, Syed Tasadaque Ali; Haberstock, Stefan; Dötsch, Volker; Bernhard, Frank; Caffrey, Martin

    2014-12-01

    Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved. PMID:25012698

  10. Development of a preprototype thermoelectric integrated membrane evaporation subsystem for water recovery

    NASA Technical Reports Server (NTRS)

    Winkler, H. E.; Roebelen, G. J., Jr.

    1980-01-01

    A three-man urine water recovery preprototype subsystem using a new concept to provide efficient potable water recovery from waste fluids on extended duration space flights has been designed, fabricated, and tested. Low power, compactness, and gravity insensitive operation are featured in this vacuum distillation subsystem that combines a hollow fiber polysulfone membrane evaporator with a thermoelectric heat pump. Application and integration of these key elements have solved problems inherent in previous reclamation subsystem designs. The hollow fiber elements provide positive liquid/gas phase control with no moving parts other than a waste liquid recirculation pump and a product water withdrawal pump. Tubular membranes provide structural integrity, improving on previous flat sheet membrane designs. A thermoelectric heat pump provides latent energy recovery.

  11. An Integrated Balanced Superconductor-Insulator-Superconductor Heterodyne Mixer on a Silicon Membrane

    NASA Astrophysics Data System (ADS)

    Westig, M. P.; Jacobs, K.; Schultz, M.; Justen, M.; Stutzki, J.; Honingh, C. E.

    2011-03-01

    We have designed and fabricated a 380-520 GHz integrated balanced NbAlAlOxNb superconductor-insulator-superconductor (SIS) heterodyne waveguide mixer for submillimeter astrophysics. The response of the mixer measured with a Fourier transform spectrometer shows excellent agreement with the design. The novelty of our device is that we deposit the complete superconducting mixer circuit (tapered slotline antennas, hybrid coupler, MIM capacitors, SIS junctions, tuning circuits and blocking filters) on top of a 9 μ m silicon membrane. The membrane is held suspended in a waveguide by 2.5 μ m thick gold plated beamleads. We will show that silicon membrane technology and a thorough device design render the integration of SIS devices with larger circuits feasible. This is an important step towards large arrays of mixers. When using an appropriate superconductor technology, these devices are scalable to higher frequencies. We will present the design, fabrication results and first results of heterodyne measurements.

  12. Membrane Compartment Occupied by Can1 (MCC) and Eisosome Subdomains of the Fungal Plasma Membrane

    PubMed Central

    Douglas, Lois M.; Wang, Hong X.; Li, Lifang; Konopka, James B.

    2011-01-01

    Studies on the budding yeast Saccharomyces cerevisiae have revealed that fungal plasma membranes are organized into different subdomains. One new domain termed MCC/eisosomes consists of stable punctate patches that are distinct from lipid rafts. The MCC/eisosome domains correspond to furrows in the plasma membrane that are about 300 nm long and 50 nm deep. The MCC portion includes integral membrane proteins, such as the tetraspanners Sur7 and Nce102. The adjacent eisosome includes proteins that are peripherally associated with the membrane, including the BAR domains proteins Pil1 and Lsp1 that are thought to promote membrane curvature. Genetic analysis of the MCC/eisosome components indicates these domains broadly affect overall plasma membrane organization. The mechanisms regulating the formation of MCC/eisosomes in model organisms will be reviewed as well as the role of these plasma membrane domains in fungal pathogenesis and response to antifungal drugs. PMID:22368779

  13. Microbead-based immunoassay using the outer membrane layer of Escherichia coli combined with autodisplayed Z-domains

    NASA Astrophysics Data System (ADS)

    Kim, Do-Hoon; Bong, Ji-Hong; Yoo, Gu; Chang, Seo-Yoon; Park, Min; Chang, Young Wook; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

    2016-01-01

    The Z-domain has the potential to control the orientation of immobilized antibodies because of its binding affinity to the Fc regions of antibodies (IgGs). In this work, Z-domains were autodisplayed on the outer membrane (OM) of Escherichia coli. OM particles were isolated and coated onto microbeads with positive, neutral, or negative surface charges. Other conditions such as incubation time and initial OM concentration were also optimized for the OM coating to obtain maximum antibody-binding. Using three kinds of model proteins with different isoelectric points (pI), streptavidin (pI = 5, negative charge at pH 7), horseradish peroxidase (pI = 7, neutral charge at pH 7), and avidin (pI = 10, positive charge at pH 7), protein immobilization onto the microbeads was carried out through physical adsorption and electrostatic interactions. Using fluorescently labeled antibodies and fluorescence-activated cell sorting, it was determined that the neutral and the positively charged microbeads effectively bound antibodies while minimizing non-specific protein binding. The OM-coated microbeads with autodisplayed Z-domains were applied to C-reactive protein immunoassay. This immunoassay achieved 5-fold improved sensitivity compared to conventional immunoassay based on physical adsorption of antibodies at the cutoff concentration of medical diagnosis of inflammatory diseases (1000 ng/ml) and cardiovascular diseases (200 ng/ml).

  14. Evolution for exogenous octanoic acid tolerance improves carboxylic acid production and membrane integrity.

    PubMed

    Royce, Liam A; Yoon, Jong Moon; Chen, Yingxi; Rickenbach, Emily; Shanks, Jacqueline V; Jarboe, Laura R

    2015-05-01

    Carboxylic acids are an attractive biorenewable chemical, but as with many biorenewables, their toxicity to microbial biocatalysts limits their fermentative production. While it is generally accepted that membrane damage is the main mechanism of fatty acid toxicity, previous metabolic engineering efforts that increased membrane integrity did not enable increased carboxylic acid production. Here we used an evolutionary approach to improve tolerance to exogenous octanoic acid, with the goal of learning design strategies from this evolved strain. This evolution of an Escherichia coli MG1655 derivative at neutral pH in minimal media produced a strain with increased tolerance not only to octanoic acid, but also to hexanoic acid, decanoic acid, n-butanol and isobutanol. This evolved strain also produced carboxylic acids at a 5-fold higher titer than its parent strain when expressing the Anaerococcus tetradius thioesterase. While it has been previously suggested that intracellular acidification may contribute to carboxylic acid toxicity, we saw no evidence that the evolved strain has increased resistance to this acidification. Characterization of the evolved strain membrane showed that it had significantly altered membrane polarization (fluidity), integrity (leakage) and composition relative to its parent. The changes in membrane composition included a significant increase in average lipid length in a variety of growth conditions, including 30°C, 42°C, carboxylic acid challenge and ethanol challenge. The evolved strain has a more dynamic membrane composition, showing both a larger number of significant changes and larger fold changes in the relative abundance of membrane lipids. These results highlight the importance of the cell membrane in increasing microbial tolerance and production of biorenewable fuels and chemicals. PMID:25839166

  15. Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains

    PubMed Central

    Veliz, Luis A.; Toro, Carlos A.; Vivar, Juan P.; Arias, Luis A.; Villegas, Jenifer; Castro, Maite A.; Brauchi, Sebastian

    2010-01-01

    Background The cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane. Methodology/Principal Findings We used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2–8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MβCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability. Conclusions/Significance These results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons. PMID:20948964

  16. INTEGRATION OF FILTRATION AND ADVANCED OXIDATION: DEVELOPMENT OF A MEMBRANE LIQUID-PHASE PLASMA REACTOR

    EPA Science Inventory

    A tiered approach will be undertaken to achieve the overall project goal of demonstrating the integrated membrane/plasma process as an innovative, affordable, sustainable and effective treatment technology for small treatment systems. The team will first use a regimented ap...

  17. Synaptotagmin-Like Proteins Control Formation of a Single Apical Membrane Domain in Epithelial Cells

    PubMed Central

    Gálvez-Santisteban, Manuel; Rodriguez-Fraticelli, Alejo E.; Bryant, David M.; Vergarajauregui, Silvia; Yasuda, Takao; Bañón-Rodríguez, Inmaculada; Bernascone, Ilenia; Datta, Anirban; Spivak, Natalie; Young, Kitty; Slim, Christiaan L.; Brakeman, Paul R.; Fukuda, Mitsunori; Mostov, Keith E.; Martín-Belmonte, Fernando

    2012-01-01

    SUMMARY The formation of epithelial tissues requires both the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. During de novo lumen formation, vectorial membrane transport contributes to formation of a singular apical membrane, resulting in contribution of each cell to only a single lumen. Here, from a functional screen for genes required for 3D epithelial architecture we identify key roles for Synaptotagmin-like proteins 2-a and 4-a (Slp2-a/4-a) in generation of a single apical surface per cell. Slp2-a localizes to the luminal membrane in a PI(4,5)P2-dependent manner, where it targets Rab27-loaded vesicles to initiate a single lumen. Vesicle tethering and fusion is controlled by Slp4-a, in conjunction with Rab27/Rab3/Rab8 and the SNARE Syntaxin-3. Together, Slp2-a/4-a co-ordinate the spatiotemporal organization of vectorial apical transport to ensure only a single apical surface, and thus formation of a single lumen, occurs per cell. PMID:22820376

  18. Proliferation and Morphogenesis of the Endoplasmic Reticulum Driven by the Membrane Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Plant Cells1[OPEN

    PubMed Central

    Ferrero, Sergi; Grados-Torrez, Ricardo Enrique; Antolín-Llovera, Meritxell; López-Iglesias, Carmen; Cortadellas, Nuria; Ferrer, Joan Carles

    2015-01-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells. PMID:26015445

  19. Proliferation and Morphogenesis of the Endoplasmic Reticulum Driven by the Membrane Domain of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase in Plant Cells.

    PubMed

    Ferrero, Sergi; Grados-Torrez, Ricardo Enrique; Leivar, Pablo; Antolín-Llovera, Meritxell; López-Iglesias, Carmen; Cortadellas, Nuria; Ferrer, Joan Carles; Campos, Narciso

    2015-07-01

    The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) has a key regulatory role in the mevalonate pathway for isoprenoid biosynthesis and is composed of an endoplasmic reticulum (ER)-anchoring membrane domain with low sequence similarity among eukaryotic kingdoms and a conserved cytosolic catalytic domain. Organized smooth endoplasmic reticulum (OSER) structures are common formations of hypertrophied tightly packed ER membranes devoted to specific biosynthetic and secretory functions, the biogenesis of which remains largely unexplored. We show that the membrane domain of plant HMGR suffices to trigger ER proliferation and OSER biogenesis. The proliferating membranes become highly enriched in HMGR protein, but they do not accumulate sterols, indicating a morphogenetic rather than a metabolic role for HMGR. The N-terminal MDVRRRPP motif present in most plant HMGR isoforms is not required for retention in the ER, which was previously proposed, but functions as an ER morphogenic signal. Plant OSER structures are morphologically similar to those of animal cells, emerge from tripartite ER junctions, and mainly build up beside the nuclear envelope, indicating conserved OSER biogenesis in high eukaryotes. Factors other than the OSER-inducing HMGR construct mediate the tight apposition of the proliferating membranes, implying separate ER proliferation and membrane association steps. Overexpression of the membrane domain of Arabidopsis (Arabidopsis thaliana) HMGR leads to ER hypertrophy in every tested cell type and plant species, whereas the knockout of the HMG1 gene from Arabidopsis, encoding its major HMGR isoform, causes ER aggregation at the nuclear envelope. Our results show that the membrane domain of HMGR contributes to ER morphogenesis in plant cells. PMID:26015445

  20. The Transmembrane Domains of β and IX Subunits Mediate the Localization of the Platelet Glycoprotein Ib-IX Complex to the Glycosphingolipid-enriched Membrane Domain.

    PubMed

    Xu, Guofeng; Shang, Dan; Zhang, Zuping; Shaw, Tanner S; Ran, Yali; López, José A; Peng, Yuandong

    2015-09-01

    We have previously reported that the structural elements of the GP Ib-IX complex required for its localization to glycosphingolipid-enriched membranes (GEMs) reside in the Ibβ and IX subunits. To identify them, we generated a series of cell lines expressing mutant GP Ibβ and GP IX where 1) the cytoplasmic tails (CTs) of either or both GP Ibβ and IX are truncated, and 2) the transmembrane domains (TMDs) of GP Ibβ and GP IX were swapped with the TMD of a non-GEMs associating molecule, human transferrin receptor. Sucrose density fractionation analysis showed that the removal of either or both of the CTs from GP Ibβ and GP IX does not alter GP Ibα-GEMs association when compared with the wild type. In contrast, swapping of the TMDs of either GP Ibβ or GP IX with that of transferrin receptor results in a significant loss (∼ 50%) of GP Ibα from the low density GEMs fractions, with the largest effect seen in the dual TMD-replaced cells (> 80% loss) when compared with the wild type cells (100% of GP Ibα present in the GEMs fractions). Under high shear flow, the TMD-swapped cells adhere poorly to a von Willebrand factor-immobilized surface to a much lesser extent than the previously reported disulfide linkage dysfunctional GP Ibα-expressing cells. Thus, our data demonstrate that the bundle of GP Ibβ and GP IX TMDs instead of their individual CTs is the structural element that mediates the β/IX complex localization to the membrane GEMs, which through the α/β disulfide linkage brings GP Ibα into the GEMs. PMID:26203189

  1. Aquaporin-0 Targets Interlocking Domains to Control the Integrity and Transparency of the Eye Lens

    PubMed Central

    Lo, Woo-Kuen; Biswas, Sondip K.; Brako, Lawrence; Shiels, Alan; Gu, Sumin; Jiang, Jean X.

    2014-01-01

    Purpose. Lens fiber cell membranes contain aquaporin-0 (AQP0), which constitutes approximately 50% of the total fiber cell membrane proteins and has a dual function as a water channel protein and an adhesion molecule. Fiber cell membranes also develop an elaborate interlocking system that is required for maintaining structural order, stability, and lens transparency. Herein, we used an AQP0-deficient mouse model to investigate an unconventional adhesion role of AQP0 in maintaining a normal structure of lens interlocking protrusions. Methods. The loss of AQP0 in AQP0−/− lens fibers was verified by Western blot and immunofluorescence analyses. Changes in membrane surface structures of wild-type and AQP0−/− lenses at age 3 to 12 weeks were examined with scanning electron microscopy. Preferential distribution of AQP0 in wild-type fiber cell membranes was analyzed with immunofluorescence and immunogold labeling using freeze-fracturing transmission electron microscopy. Results. Interlocking protrusions in young differentiating fiber cells developed normally but showed minor abnormalities at approximately 50 μm deep in the absence of AQP0 in all ages studied. Strikingly, protrusions in maturing fiber cells specifically underwent uncontrolled elongation, deformation, and fragmentation, while cells still retained their overall shape. Later in the process, these changes eventually resulted in fiber cell separation, breakdown, and cataract formation in the lens core. Immunolabeling at the light microscopy and transmission electron microscopy levels demonstrated that AQP0 was particularly enriched in interlocking protrusions in wild-type lenses. Conclusions. This study suggests that AQP0 exerts its primary adhesion or suppression role specifically to maintain the normal structure of interlocking protrusions that is critical to the integrity and transparency of the lens. PMID:24458158

  2. A combined integrating and differentiating matrix formulation for boundary value problems on rectangular domains

    NASA Technical Reports Server (NTRS)

    Lakin, W. D.

    1986-01-01

    Integrating and differentiating matrices allow the numerical integration and differential of functions whose values are known at points of a discrete grid. Previous derivations of these matrices were restricted to one dimensional grids or to rectangular grids with uniform spacing in at least one direction. Integrating and differentiating matrices were developed for grids with nonuniform spacing in both directions. The use of these matrices as operators to reformulate boundary value problems on rectangular domains as matrix problems for a finite dimensional solution vector is considered. The method requires nonuniform grids which include near boundary points. An eigenvalue problem for the transverse vibrations of a simply supported rectangular plate is solved to illustrate the method.

  3. SAS-1 Is a C2 Domain Protein Critical for Centriole Integrity in C. elegans

    PubMed Central

    Delattre, Marie; Balestra, Fernando R.; Blanchoud, Simon; Finger, Susanne; Knott, Graham; Müller-Reichert, Thomas; Gönczy, Pierre

    2014-01-01

    Centrioles are microtubule-based organelles important for the formation of cilia, flagella and centrosomes. Despite progress in understanding the underlying assembly mechanisms, how centriole integrity is ensured is incompletely understood, including in sperm cells, where such integrity is particularly critical. We identified C. elegans sas-1 in a genetic screen as a locus required for bipolar spindle assembly in the early embryo. Our analysis reveals that sperm-derived sas-1 mutant centrioles lose their integrity shortly after fertilization, and that a related defect occurs when maternal sas-1 function is lacking. We establish that sas-1 encodes a C2 domain containing protein that localizes to centrioles in C. elegans, and which can bind and stabilize microtubules when expressed in human cells. Moreover, we uncover that SAS-1 is related to C2CD3, a protein required for complete centriole formation in human cells and affected in a type of oral-facial-digital (OFD) syndrome. PMID:25412110

  4. Simulation of planar integrated photonics devices with the LLNL time- domain finite-difference code suite

    SciTech Connect

    McLeod, R.; Hawkins, R.J.; Kallman, J.S.

    1991-04-01

    Interest has recently grown in applying microwave modeling techniques to optical circuit modeling. One of the simplest, yet most powerful, microwave simulation techniques is the finite-difference time-domain algorithm (FDTD). In this technique, the differential form of the time-domain Maxwell's equations are discretized and all derivatives are approximated as differences. Minor algebraic manipulations on the resulting equations produces a set of update equations that produce fields at a given time step from fields at the previous time step. The FDTD algorithm, then, is quite simple. Source fields are launched into the discrete grid by some means. The FDTD equations advance these fields in time. At the boundaries of the grid, special update equations called radiation conditions are applied that approximate a continuing, infinite space. Because virtually no assumptions are made in the development of the FDTD method, the algorithm is able to represent a wide-range of physical effects. Waves can propagate in any direction, multiple reflections within structures can cause resonances, multiple modes of various polarizations can be launched, each of which may generate within the device an infinite spectrum of bound and radiation modes. The ability to model these types of general physical effects is what makes the FDTD method interesting to the field of optics. In this paper, we discuss the application of the finite-difference time-domain technique to integrated optics. Animations will be shown of the simulations of a TE coupler, TM grating, and a TE integrated detector. 3 refs., 1 fig.

  5. Silymarin protects plasma membrane and acrosome integrity in sperm treated with sodium arsenite

    PubMed Central

    Eskandari, Farzaneh; Momeni, Hamid Reza

    2016-01-01

    Background: Exposure to arsenic is associated with impairment of male reproductive function by inducing oxidative stress. Silymarin with an antioxidant property scavenges free radicals. Objective: The aim of this study was to investigate if silymarin can prevent the adverse effects of sodium arsenite on ram sperm plasma membrane and acrosome integrity. Materials and Methods: Ram epidydimal spermatozoa were divided into five groups: spermatozoa at 0 hr, spermatozoa at 180 min (control), spermatozoa treated with silymarin (20 μM) + sodium arsenite (10 μM) for 180 min, spermatozoa treated with sodium arsenite (10 μM) for 180 min and spermatozoa treated with silymarin (20 μM) for 180 min. Double staining of Hoechst and propidium iodide was performed to evaluate sperm plasma membrane integrity, whereas comassie brilliant blue staining was used to assess acrosome integrity. Results: Plasma membrane (p< 0.001) and acrosome integrity (p< 0.05) of the spermatozoa were significantly reduced in sodium arsenite group compared to the control. In silymarin + sodium arsenite group, silymarin was able to significantly (p< 0.001) ameliorate the adverse effects of sodium arsenite on these sperm parameters compared to sodium arsenite group. The incubation of sperm for 180 min (control group) showed a significant (p< 0.001) decrease in acrosome integrity compared to the spermatozoa at 0 hour. The application of silymarin alone for 180 min could also significantly (p< 0.05) increase sperm acrosome integrity compared to the control. Conclusion: Silymarin as a potent antioxidant could compensate the adverse effects of sodium arsenite on the ram sperm plasma membrane and acrosome integrity. PMID:27141548

  6. The Major Outer Sheath Protein (Msp) of Treponema denticola Has a Bipartite Domain Architecture and Exists as Periplasmic and Outer Membrane-Spanning Conformers

    PubMed Central

    Anand, Arvind; Luthra, Amit; Edmond, Maxwell E.; Ledoyt, Morgan; Caimano, Melissa J.

    2013-01-01

    The major outer sheath protein (Msp) is a primary virulence determinant in Treponema denticola, as well as the parental ortholog for the Treponema pallidum repeat (Tpr) family in the syphilis spirochete. The Conserved Domain Database (CDD) server revealed that Msp contains two conserved domains, major outer sheath proteinN (MOSPN) and MOSPC, spanning residues 77 to 286 and 332 to 543, respectively, within the N- and C-terminal regions of the protein. Circular dichroism (CD) spectroscopy, Triton X-114 (TX-114) phase partitioning, and liposome incorporation demonstrated that full-length, recombinant Msp (MspFl) and a recombinant protein containing MOSPC, but not MOSPN, form amphiphilic, β-sheet-rich structures with channel-forming activity. Immunofluorescence analysis of intact T. denticola revealed that only MOSPC contains surface-exposed epitopes. Data obtained using proteinase K accessibility, TX-114 phase partitioning, and cell fractionation revealed that Msp exists as distinct OM-integrated and periplasmic trimers. MspFl folded in Tris buffer contained slightly less β-sheet structure than detergent-folded MspFl; both forms, however, partitioned into the TX-114 detergent-enriched phase. CDD analysis of the nine Tpr paralogs predicted to be outer membrane proteins (OMPs) revealed that seven have an Msp-like bipartite structure; phylogenetic analysis revealed that the MOSPN and MOSPC domains of Msp are most closely related to those of TprK. Based upon our collective results, we propose a model whereby a newly exported, partially folded intermediate can be either processed for OM insertion by the β-barrel assembly machinery (BAM) or remain periplasmic, ultimately forming a stable, water-soluble trimer. Extrapolated to T. pallidum, our model enables us to explain how individual Tprs can localize to either the periplasmic (e.g., TprK) or OM (e.g., TprC) compartments. PMID:23457251

  7. Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity*

    PubMed Central

    Golczak, Marcin; Kiser, Philip D.; Lodowski, David T.; Maeda, Akiko; Palczewski, Krzysztof

    2010-01-01

    Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A2 treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid

  8. A yeast toxic mutant of HET-s amyloid disrupts membrane integrity.

    PubMed

    Ta, Ha Phuong; Berthelot, Karine; Coulary-Salin, Bénédicte; Castano, Sabine; Desbat, Bernard; Bonnafous, Pierre; Lambert, Olivier; Alves, Isabel; Cullin, Christophe; Lecomte, Sophie

    2012-09-01

    Many studies have pointed out the interaction between amyloids and membranes, and their potential involvement in amyloid toxicity. Previously, we generated a yeast toxic amyloid mutant (M8) from the harmless amyloid protein by changing a few residues of the Prion Forming Domain of HET-s (PFD HET-s(218-289)) and clearly demonstrated the complete different behaviors of the non-toxic Wild Type (WT) and toxic amyloid (called M8) in terms of fiber morphology, aggregation kinetics and secondary structure. In this study, we compared the interaction of both proteins (WT and M8) with membrane models, as liposomes or supported bilayers. We first demonstrated that the toxic protein (M8) induces a significant leakage of liposomes formed with negatively charged lipids and promotes the formation of microdomains inside the lipid bilayer (as potential "amyloid raft"), whereas the non-toxic amyloid (WT) only binds to the membrane without further perturbations. The secondary structure of both amyloids interacting with membrane is preserved, but the anti-symmetric PO(2)(-) vibration is strongly shifted in the presence of M8. Secondly, we established that the presence of membrane models catalyzes the amyloidogenesis of both proteins. Cryo-TEM (cryo-transmission electron microscopy) images show the formation of long HET-s fibers attached to liposomes, whereas a large aggregation of the toxic M8 seems to promote a membrane disruption. This study allows us to conclude that the toxicity of the M8 mutant could be due to its high propensity to interact and disrupt lipid membranes. PMID:22562024

  9. Importance of Membrane Structural Integrity for RPE65 Retinoid Isomerization Activity

    SciTech Connect

    Golczak, Marcin; Kiser, Philip D.; Lodowski, David T.; Maeda, Akiko; Palczewski, Krzysztof

    2010-04-05

    Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A{sup 2} treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and

  10. Push-pull integrated-optics Mach-Zehnder interferometer with domain inversion in one branch

    NASA Astrophysics Data System (ADS)

    Jaeger, Nicolas A. F.; Huang, Lisheng

    1995-02-01

    We report on a push-pull integrated-optics Mach-Zehnder interferometer having one domain-inverted branch and one noninverted branch. It is fabricated in the c+ side of a LiNbO3 substrate. We achieve ferroelectric domain inversion in one branch of the waveguide by doping it more heavily with Ti than the other branch and by using a high-temperature treatment that raises the temperature above the doped Curie temperature of the one branch but not of the other. The cumulative electro-optic effect of the inverted branch is found to be opposite that in the noninverted branch when both are subjected to the same electric field.

  11. A hybrid method for solving time-domain integral equations in transient scattering

    NASA Astrophysics Data System (ADS)

    Tijhuis, A. G.; Wiemans, R.; Kuester, E. F.

    A new hybrid method is proposed for the numerical solution of integral equations describing transient scattering problems. The basic idea behind the method is to first discretize in space, and then solve the resulting system of linear time-domain equations by carrying out a temporal Laplace transformation. This approach combines the efficiency of the marching-on-in-time method with the stability and the accuracy of frequency-domain techniques. Numerical results are presented for the scattering of E-polarized, pulsed waves by a one-dimensionally inhomogeneous, lossy dielectric slab located in between two homogeneous, lossless dielectric half-spaces, and by a radially inhomogeneous, lossy dielectric circular cylinder embedded in vacuum. For these problems, the hybrid method turns out to be more powerful than existing solution techniques.

  12. Association of lipids with integral membrane surface proteins of Mycoplasma hyorhinis

    SciTech Connect

    Bricker, T.M.; Boyer, M.J.; Keith, J.; Watson-McKown, R.; Wise, K.S.

    1988-02-01

    Triton X-114 (TX-114)-phase fractionation was used to identify and characterize integral membrane surface proteins of the wall-less procaryote Mycoplasma hyorhinis GDL. Phase fractionation of mycoplasmas followed by analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed selective partitioning of approximately 30 (/sup 35/S)methionine-labeled intrinsic membrane proteins into the TX-114 phase. Similar analysis of (/sup 3/H)palmitate-labeled cells showed that approximately 20 proteins of this organism were associated with lipid, all of which also efficiently partitioned as integral membrane components into the detergent phase. Immunoblotting and immunoprecipitation of TX-114-phase proteins from /sup 125/I-surface-labeled cells with four monoclonal antibodies to distinct surface epitopes of M. hyorhinis identified surface proteins p120, p70, p42, and p23 as intrinsic membrane components. Immunoprecipitation of (/sup 3/H)palmitate-labeled TX-114-phase proteins further established that surface proteins p120, p70, and p23 (a molecule that mediates complement-dependent mycoplasmacidal monoclonal antibody activity) were among the lipid-associated proteins of this organism. Two of these proteins, p120 and p123, were acidic (pI less than or equal to 4.5), as shown by two-dimensional isoelectric focusing. This study established that M. hyorhinis contains an abundance of integral membrane proteins tightly associated with lipids and that many of these proteins are exposed at the external surface of the single limiting plasma membrane. Monoclonal antibodies are reported that will allow detailed analysis of the structure and processing of lipid-associated mycoplasma proteins.

  13. Recovery of Flavonoids from Orange Press Liquor by an Integrated Membrane Process

    PubMed Central

    Cassano, Alfredo; Conidi, Carmela; Ruby-Figueroa, René

    2014-01-01

    Orange press liquor is a by-product generated by the citrus processing industry containing huge amounts of natural phenolic compounds with recognized antioxidant activity. In this work, an integrated membrane process for the recovery of flavonoids from orange press liquors was investigated on a laboratory scale. The liquor was previously clarified by ultrafiltration (UF) in selected operating conditions by using hollow fiber polysulfone membranes. Then, the clarified liquor with a total soluble solids (TSS) content of 10 g·100 g−1 was pre-concentrated by nanofiltration (NF) up to 32 g TSS 100 g−1 by using a polyethersulfone spiral-wound membrane. A final concentration step, up to 47 g TSS 100 g−1, was performed by using an osmotic distillation (OD) apparatus equipped with polypropylene hollow fiber membranes. Suspended solids were completely removed in the UF step producing a clarified liquor containing most part of the flavonoids of the original press liquor due to the low rejection of the UF membrane towards these compounds. Flavanones and anthocyanins were highly rejected by the NF membrane, producing a permeate stream with a TSS content of 4.5 g·100 g−1. An increasing of both the flavanones and anthocyanins concentration was observed in the NF retentate by increasing the volume reduction factor (VRF). The final concentration of flavonoids by OD produced a concentrated solution of interest for nutraceutical and pharmaceutical applications. PMID:25116725

  14. Multichannel detection of ionic currents through two nanopores fabricated on integrated Si3N4 membranes.

    PubMed

    Yanagi, Itaru; Akahori, Rena; Aoki, Mayu; Harada, Kunio; Takeda, Ken-Ichi

    2016-08-16

    Integration of solid-state nanopores and multichannel detection of signals from each nanopore are effective measures for realizing high-throughput nanopore sensors. In the present study, we demonstrated fabrication of Si3N4 membrane arrays and the simultaneous measurement of ionic currents through two nanopores formed in two adjacent membranes. Membranes with thicknesses as low as 6.4 nm and small nanopores with diameters of less than 2 nm could be fabricated using the poly-Si sacrificial-layer process and multilevel pulse-voltage injection. Using the fabricated nanopore membranes, we successfully achieved simultaneous detection of clear ionic-current blockades when single-stranded short homopolymers (poly(dA)60) passed through two nanopores. In addition, we investigated the signal crosstalk and leakage current among separated chambers. When two nanopores were isolated on the front surface of the membrane, there was no signal crosstalk or leakage current between the chambers. However, when two nanopores were isolated on the backside of the Si substrate, signal crosstalk and leakage current were observed owing to high-capacitance coupling between the chambers and electrolysis of water on the surface of the Si substrate. The signal crosstalk and leakage current could be suppressed by oxidizing the exposed Si surface in the membrane chip. Finally, the observed ionic-current blockade when poly(dA)60 passed through the nanopore in the oxidized chip was approximately half of that observed in the non-oxidized chip. PMID:27440476

  15. A novel approach for application of nylon membranes in the biosensing domain

    NASA Astrophysics Data System (ADS)

    Farahmand, Elham; Ibrahim, Fatimah; Hosseini, Samira; Rothan, Hussin A.; Yusof, Rohana; Koole, Leo H.; Djordjevic, Ivan

    2015-10-01

    In this paper we report the polymer-coated microporous nylon membranes and their application as platforms for protein immobilization and subsequent detection of the dengue virus (DV) in blood serum. Protein recognition experiments were performed with enzyme-linked immunosorbent assay (ELISA). The polymers used for coatings were synthesized by free-radical polymerization reaction between methyl methacrylate (MMA) and methacrylic acid (MAA) in different concentrations. The MAA monomer has carefully been chosen to generate polymers with pendant carboxyl (-COOH) groups, which also exist on polymer surfaces. A high degree of control over surface-exposed -COOH groups has been achieved through variation of monomers concentration in polymerization reaction. The general aspect of this work relies on the dengue antibody (Ab) immobilization on surface -COOH groups via physical attachment or covalent immobilization. Prior to Ab immobilization and ELISA experiment, polymer-coated nylon samples were analyzed in detail for their physical properties by atomic force microscopy (AFM), scanning electron microscopy (SEM), and water-in-air contact angle (WCA) measurements. Membranes were further analyzed by Fourier transform infrared spectroscopy (FTIR) in order to establish the relationship between wettability, porosity, and surface roughness with chemical composition and concentration of -COOH groups on the coating's surface. Optimized coatings have shown high sensitivity towards dengue Ab molecules, revealing fundamental aspect of polymer-protein interfaces as a function of surface -COOH groups' concentration.

  16. The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains.

    PubMed

    Ros, Uris; Edwards, Michelle A; Epand, Raquel F; Lanio, Maria E; Schreier, Shirley; Yip, Christopher M; Alvarez, Carlos; Epand, Richard M

    2013-11-01

    Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation. PMID:23954588

  17. Towards a Conceptual Design of a Cross-Domain Integrative Information System for the Geosciences

    NASA Astrophysics Data System (ADS)

    Zaslavsky, I.; Richard, S. M.; Valentine, D. W.; Malik, T.; Gupta, A.

    2013-12-01

    As geoscientists increasingly focus on studying processes that span multiple research domains, there is an increased need for cross-domain interoperability solutions that can scale to the entire geosciences, bridging information and knowledge systems, models, software tools, as well as connecting researchers and organization. Creating a community-driven cyberinfrastructure (CI) to address the grand challenges of integrative Earth science research and education is the focus of EarthCube, a new research initiative of the U.S. National Science Foundation. We are approaching EarthCube design as a complex socio-technical system of systems, in which communication between various domain subsystems, people and organizations enables more comprehensive, data-intensive research designs and knowledge sharing. In particular, we focus on integrating 'traditional' layered CI components - including information sources, catalogs, vocabularies, services, analysis and modeling tools - with CI components supporting scholarly communication, self-organization and social networking (e.g. research profiles, Q&A systems, annotations), in a manner that follows and enhances existing patterns of data, information and knowledge exchange within and across geoscience domains. We describe an initial architecture design focused on enabling the CI to (a) provide an environment for scientifically sound information and software discovery and reuse; (b) evolve by factoring in the impact of maturing movements like linked data, 'big data', and social collaborations, as well as experience from work on large information systems in other domains; (c) handle the ever increasing volume, complexity and diversity of geoscience information; (d) incorporate new information and analytical requirements, tools, and techniques, and emerging types of earth observations and models; (e) accommodate different ideas and approaches to research and data stewardship; (f) be responsive to the existing and anticipated needs

  18. Nanowire-integrated microporous silicon membrane for continuous fluid transport in micro cooling device

    SciTech Connect

    So, Hongyun; Pisano, Albert P.; Cheng, Jim C.

    2013-10-14

    We report an efficient passive micro pump system combining the physical properties of nanowires and micropores. This nanowire-integrated microporous silicon membrane was created to feed coolant continuously onto the surface of the wick in a micro cooling device to ensure it remains hydrated and in case of dryout, allow for regeneration of the system. The membrane was fabricated by photoelectrochemical etching to form micropores followed by hydrothermal growth of nanowires. This study shows a promising approach to address thermal management challenges for next generation electronic devices with absence of external power.

  19. Single Mutations in the Transmembrane Domains of Maize Plasma Membrane Aquaporins Affect the Activity of Monomers within a Heterotetramer.

    PubMed

    Berny, Marie C; Gilis, Dimitri; Rooman, Marianne; Chaumont, François

    2016-07-01

    Aquaporins are channels facilitating the diffusion of water and/or small uncharged solutes across biological membranes. They assemble as homotetramers but some of them also form heterotetramers, especially in plants. In Zea mays, aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily are clustered into two groups, PIP1 and PIP2, which exhibit different water-channel activities when expressed in Xenopus oocytes. When PIP1 and PIP2 isoforms are co-expressed, they physically interact to modulate their subcellular localization and channel activity. Here, we demonstrated by affinity chromatography purification that, when co-expressed in Xenopus oocytes, the maize PIP1;2 and PIP2;5 isoforms assemble as homo- and heterodimers within heterotetramers. We built the 3D structure of such heterotetramers by comparative modeling on the basis of the spinach SoPIP2;1 X-ray structure and identified amino acid residues in the transmembrane domains which putatively interact at the interfaces between monomers. Their roles in the water-channel activity, subcellular localization, protein abundance, and physical interaction were investigated by mutagenesis. We highlighted single-residue substitutions that either inactivated PIP2;5 or activated PIP1;2 without affecting their interaction. Interestingly, the Phe220Ala mutation in the transmembrane domain 5 of PIP1;2 activated its water-channel activity and, at the same time, inactivated PIP2;5 within a heterotetramer. Altogether, these data contribute to a better understanding of the interaction mechanisms between PIP isoforms and the role of heterotetramerization on their water-channel activity. PMID:27109604

  20. Studies on improved integrated membrane-based chromatographic process for bioseparation

    NASA Astrophysics Data System (ADS)

    Xu, Yanke

    To improve protein separation and purification directly from a fermentation broth, a novel membrane filtration-cum-chromatography device configuration having a relatively impermeable coated zone near the hollow fiber module outlet has been developed. The integrated membrane filtration-cum-chromatography unit packed with chromatographic beads on the shell side of the hollow fiber unit enjoys the advantages of both membrane filtration and chromatography; it allows one to load the chromatographic media directly from t