TFEB activation promotes the recruitment of lysosomal glycohydrolases β-hexosaminidase and β-galactosidase to the plasma membrane

DOE Office of Scientific and Technical Information (OSTI.GOV)

Magini, Alessandro; Department of Medical and Biological Sciences; Polchi, Alice

2013-10-18

Highlights: •TFEB activation promotes the increase of Hex and Gal activities. •The increase of Hex and Gal activities is related to transcriptional regulation. •TFEB promotes the recruitment of mature Hex and Gal on cell surface. -- Abstract: Lysosomes are membrane-enclosed organelles containing acid hydrolases. They mediate a variety of physiological processes, such as cellular clearance, lipid homeostasis, energy metabolism and pathogen defence. Lysosomes can secrete their content through a process called lysosome exocytosis in which lysosomes fuse with the plasma membrane realising their content into the extracellular milieu. Lysosomal exocytosis is not only responsible for the secretion of lysosomal enzymes,more » but it also has a crucial role in the plasma membrane repair. Recently, it has been demonstrated that lysosome response to the physiologic signals is regulated by the transcription factor EB (TFEB). In particular, lysosomal secretion is transcriptionally regulated by TFEB which induces both the docking and fusion of lysosomes with the plasma membrane. In this work we demonstrated that TFEB nuclear translocation is accompanied by an increase of mature glycohydrolases β-hexosaminidase and β-galactosidase on cell surface. This evidence contributes to elucidate an unknown TFEB biological function leading the lysosomal glycohydrolases on plasma membrane.« less

A PI4P-driven electrostatic field controls cell membrane identity and signaling in plants

PubMed Central

Simon, Mathilde Laetitia Audrey; Platre, Matthieu Pierre; Marquès-Bueno, Maria Mar; Armengot, Laia; Stanislas, Thomas; Bayle, Vincent; Caillaud, Marie-Cécile; Jaillais, Yvon

2016-01-01

Many signaling proteins permanently or transiently localize to specific organelles for function. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PI4P). Our results further reveal that, contrarily to other eukaryotes, PI4P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID, as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) family, which are involved in brassinosteroid and receptor-like kinase signaling. We anticipate that this PI4P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition. PMID:27322096

A PtdIns(4)P-driven electrostatic field controls cell membrane identity and signalling in plants.

PubMed

Simon, Mathilde Laetitia Audrey; Platre, Matthieu Pierre; Marquès-Bueno, Maria Mar; Armengot, Laia; Stanislas, Thomas; Bayle, Vincent; Caillaud, Marie-Cécile; Jaillais, Yvon

2016-06-20

Many signalling proteins permanently or transiently localize to specific organelles. It is well established that certain lipids act as biochemical landmarks to specify compartment identity. However, they also influence membrane biophysical properties, which emerge as important features in specifying cellular territories. Such parameters include the membrane inner surface potential, which varies according to the lipid composition of each organelle. Here, we found that the plant plasma membrane (PM) and the cell plate of dividing cells have a unique electrostatic signature controlled by phosphatidylinositol-4-phosphate (PtdIns(4)P). Our results further reveal that, contrarily to other eukaryotes, PtdIns(4)P massively accumulates at the PM, establishing it as a critical hallmark of this membrane in plants. Membrane surface charges control the PM localization and function of the polar auxin transport regulator PINOID as well as proteins from the BRI1 KINASE INHIBITOR1 (BKI1)/MEMBRANE ASSOCIATED KINASE REGULATOR (MAKR) family, which are involved in brassinosteroid and receptor-like kinase signalling. We anticipate that this PtdIns(4)P-driven physical membrane property will control the localization and function of many proteins involved in development, reproduction, immunity and nutrition.

Determinants in the β and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor.

PubMed

Rudell, Jolene Chang; Borges, Lucia S; Rudell, John B; Beck, Kenneth A; Ferns, Michael J

2014-01-03

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.

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

PubMed

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

2017-10-27

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

BAR domain proteins regulate Rho GTPase signaling.

PubMed

Aspenström, Pontus

2014-01-01

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

Effect of therapeutic concentration of lithium on live HEK293 cells; increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane.

PubMed

Vosahlikova, Miroslava; Ujcikova, Hana; Chernyavskiy, Oleksandr; Brejchova, Jana; Roubalova, Lenka; Alda, Martin; Svoboda, Petr

2017-05-01

The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1mM LiCl for 7 or 21days. The alteration of Na + /K + -ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells. Na + /K + -ATPase level was determined by [ 3 H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan. Na + /K + -ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells. Prolonged exposure of HEK293 cells to 1mM LiCl results in up-regulation of Na + /K + -ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes. Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na + /K + -ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

Caveolae as plasma membrane sensors, protectors and organizers.

PubMed

Parton, Robert G; del Pozo, Miguel A

2013-02-01

Caveolae are submicroscopic, plasma membrane pits that are abundant in many mammalian cell types. The past few years have seen a quantum leap in our understanding of the formation, dynamics and functions of these enigmatic structures. Caveolae have now emerged as vital plasma membrane sensors that can respond to plasma membrane stresses and remodel the extracellular environment. Caveolae at the plasma membrane can be removed by endocytosis to regulate their surface density or can be disassembled and their structural components degraded. Coat proteins, called cavins, work together with caveolins to regulate the formation of caveolae but also have the potential to dynamically transmit signals that originate in caveolae to various cellular destinations. The importance of caveolae as protective elements in the plasma membrane, and as membrane organizers and sensors, is highlighted by links between caveolae dysfunction and human diseases, including muscular dystrophies and cancer.

The Role of Water Distribution Controlled by Transmembrane Potentials in the Cytochrome c-Cardiolipin Interaction: Revealing from Surface-Enhanced Infrared Absorption Spectroscopy.

PubMed

Zeng, Li; Wu, Lie; Liu, Li; Jiang, Xiue

2017-11-02

The interaction of cytochrome c (cyt c) with cardiolipin (CL) plays a crucial role in apoptotic functions, however, the changes of the transmembrane potential in governing the protein behavior at the membrane-water interface have not been studied due to the difficulties in simultaneously monitoring the interaction and regulating the electric field. Herein, surface-enhanced infrared absorption (SEIRA) spectroelectrochemistry is employed to study the mechanism of how the transmembrane potentials control the interaction of cyt c with CL membranes by regulating the electrode potentials of an Au film. When the transmembrane potential decreases, the water content at the interface of the membranes can be increased to slow down protein adsorption through decreasing the hydrogen-bond and hydrophobic interactions, but regulates the redox behavior of CL-bound cyt c through a possible water-facilitated proton-coupled electron transfer process. Our results suggest that the potential drop-induced restructure of the CL conformation and the hydration state could modify the structure and function of CL-bound cyt c on the lipid membrane. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The plasma membrane: Penultimate regulator of ADAM sheddase function.

PubMed

Reiss, Karina; Bhakdi, Sucharit

2017-11-01

ADAM10 and ADAM17 are the best characterized members of the ADAM (A Disintegrin and Metalloproteinase) - family of transmembrane proteases. Both are involved diverse physiological and pathophysiological processes. ADAMs are known to be regulated by posttranslational mechanisms. However, emerging evidence indicates that the plasma membrane with its unique dynamic properties may additionally play an important role in controlling sheddase function. Membrane events that could contribute to regulation of ADAM-function are summarized. Surface expression of peptidolytic activity should be differentiated from ADAM-sheddase function since the latter additionally requires that the protease finds its substrate in the lipid bilayer. We propose that this is achieved through horizontal and vertical reorganization of membrane nanoarchitecture coordinately occurring at the sites of sheddase activation. Reshuffling of nanodomains thereby guides traffic of enzyme and substrate to each other. For ADAM17 phosphatidylserine exposure is required to then induce its shedding function. The novel concept that physicochemical properties of the lipid bilayer govern the action of ADAM-proteases may be extendable to other functional proteins that act at the cell surface. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. Copyright © 2017. Published by Elsevier B.V.

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane

PubMed Central

Dittman, Jeremy S.; Kaplan, Joshua M.

2006-01-01

After synaptic vesicle fusion, vesicle proteins must be segregated from plasma membrane proteins and recycled to maintain a functional vesicle pool. We monitored the distribution of synaptobrevin, a vesicle protein required for exocytosis, in Caenorhabditis elegans motor neurons by using a pH-sensitive synaptobrevin GFP fusion protein, synaptopHluorin. We estimated that 30% of synaptobrevin was present in the plasma membrane. By using a panel of endocytosis and exocytosis mutants, we found that the majority of surface synaptobrevin derives from fusion of synaptic vesicles and that, in steady state, synaptobrevin equilibrates throughout the axon. The surface synaptobrevin was enriched near active zones, and its spatial extent was regulated by the clathrin adaptin AP180. These results suggest that there is a plasma membrane reservoir of synaptobrevin that is supplied by the synaptic vesicle cycle and available for retrieval throughout the axon. The size of the reservoir is set by the relative rates of exo- and endocytosis. PMID:16844789

The Plasma Membrane Sialidase NEU3 Regulates the Malignancy of Renal Carcinoma Cells by Controlling β1 Integrin Internalization and Recycling*

PubMed Central

Tringali, Cristina; Lupo, Barbara; Silvestri, Ilaria; Papini, Nadia; Anastasia, Luigi; Tettamanti, Guido; Venerando, Bruno

2012-01-01

The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy. PMID:23139422

Regulation of surface expression of TRPV2 channels in the retinal pigment epithelium.

PubMed

Reichhart, Nadine; Keckeis, Susanne; Fried, Frederik; Fels, Gabriele; Strauss, Olaf

2015-06-01

The retinal pigment epithelium (RPE) interacts closely with the photoreceptors in fulfilling tasks of visual function. Since an understanding of the RPE function is essential for understanding the patho-mechanisms involved in vision loss, we explored the regulation of the vanilloid receptor subtype transient receptor potential TRPV2 channels that trigger insulin-like growth factor-1 (IGF-1)-induced vascular endothelial growth factor A (VEGF-A) secretion. Immunohistochemistry was used to assess TRPV2 expression in retinal cross-sections or ARPE-19 cells, and surface expression of TRPV2 was quantified using confocal microscopy. Membrane currents of ARPE-19 cells were recorded using a whole-cell configuration of the patch-clamp technique. TRPV2 expression was detected in the RPE of the mouse retina as well as in ARPE-19 cells. Increasing the temperature to 45 °C activated membrane conductance sensitive to SKF-96365 and ruthenium red in 60 % of cells. Preincubation with either cannabidiol (CBD) or IGF-1 led to a three- or fourfold increase in current density, respectively, in all cells, which was blocked by SKF-96365. In contrast to IGF-1, CBD stimulation of membrane conductance was further increased by heat. TRPV2 surface expression was increased by both IGF-1 and CBD, with the increase by CBD twice as large as that by IGF-1. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished the effects on membrane conductance and surface expression. Both CBD and IGF-1 enhance TRPV2 channel activity by specific proportions of both channel activation and PI 3-kinase-dependent surface expression: IGF-1 predominantly increases ion channel activity, whereas CBD is more active in increasing TRPV2 surface expression. Thus, differential regulation of TRPV2 surface expression is an important mechanism for modulating the responsiveness of the RPE to growth factors.

Regulation of AMPA receptor localization in lipid rafts

PubMed Central

Hou, Qingming; Huang, Yunfei; Amato, Stephen; Snyder, Solomon H.; Huganir, Richard L.; Man, Heng-Ye

2009-01-01

Lipid rafts are special microdomains enriched in cholesterol, sphingolipids and certain proteins, and play important roles in a variety of cellular functions including signal transduction and protein trafficking. We report that in cultured cortical and hippocampal neurons the distribution of lipid rafts is development-dependent. Lipid rafts in mature neurons exist on the entire cell-surface and display a high degree of mobility. AMPA receptors co-localize and associate with lipid rafts in the plasma membrane. The association of AMPARs with rafts is under regulation; through the NOS–NO pathway, NMDA receptor activity increases AMPAR localization in rafts. During membrane targeting, AMPARs insert into or at close proximity of the surface raft domains. Perturbation of lipid rafts dramatically suppresses AMPA receptor exocytosis, resulting in significant reduction in AMPAR cell-surface expression. PMID:18411055

LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

PubMed

Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

2010-12-03

LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

Regulation of Cl^- Channels in Normal and Cystic Fibrosis Airway Epithelial Cells by Extracellular ATP

NASA Astrophysics Data System (ADS)

Stutts, M. J.; Chinet, T. C.; Mason, S. J.; Fullton, J. M.; Clarke, L. L.; Boucher, R. C.

1992-03-01

The rate of Cl^- secretion by human airway epithelium is determined, in part, by apical cell membrane Cl^- conductance. In cystic fibrosis airway epithelia, defective regulation of Cl^- conductance decreases the capability to secrete Cl^-. Here we report that extracytosolic ATP in the luminal bath of cultured human airway epithelia increased transepithelial Cl^- secretion and apical membrane Cl^- permeability. Single-channel studies in excised membrane patches revealed that ATP increased the open probability of outward rectifying Cl^- channels. The latter effect occurs through a receptor mechanism that requires no identified soluble second messengers and is insensitive to probes of G protein function. These results demonstrate a mode of regulation of anion channels by binding ATP at the extracellular surface. Regulation of Cl^- conductance by external ATP is preserved in cystic fibrosis airway epithelia.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane

PubMed Central

Salanenka, Yuliya; Verstraeten, Inge; Löfke, Christian; Tabata, Kaori; Naramoto, Satoshi; Glanc, Matouš; Friml, Jiří

2018-01-01

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses. PMID:29463731

Regulation of cell protrusions by small GTPases during fusion of the neural folds

PubMed Central

Rolo, Ana; Savery, Dawn; Escuin, Sarah; de Castro, Sandra C; Armer, Hannah EJ; Munro, Peter MG; Molè, Matteo A; Greene, Nicholas DE; Copp, Andrew J

2016-01-01

Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development. DOI: http://dx.doi.org/10.7554/eLife.13273.001 PMID:27114066

Engineering Lipid Bilayer Membranes for Protein Studies

PubMed Central

Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton

2013-01-01

Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques. PMID:24185908

Loss of electrostatic cell-surface repulsion mediates myelin membrane adhesion and compaction in the central nervous system.

PubMed

Bakhti, Mostafa; Snaidero, Nicolas; Schneider, David; Aggarwal, Shweta; Möbius, Wiebke; Janshoff, Andreas; Eckhardt, Matthias; Nave, Klaus-Armin; Simons, Mikael

2013-02-19

During the development of the central nervous system (CNS), oligodendrocytes wrap their plasma membrane around axons to form a multilayered stack of tightly attached membranes. Although intracellular myelin compaction and the role of myelin basic protein has been investigated, the forces that mediate the close interaction of myelin membranes at their external surfaces are poorly understood. Such extensive bilayer-bilayer interactions are usually prevented by repulsive forces generated by the glycocalyx, a dense and confluent layer of large and negatively charged oligosaccharides. Here we investigate the molecular mechanisms underlying myelin adhesion and compaction in the CNS. We revisit the role of the proteolipid protein and analyze the contribution of oligosaccharides using cellular assays, biophysical tools, and transgenic mice. We observe that differentiation of oligodendrocytes is accompanied by a striking down-regulation of components of their glycocalyx. Both in vitro and in vivo experiments indicate that the adhesive properties of the proteolipid protein, along with the reduction of sialic acid residues from the cell surface, orchestrate myelin membrane adhesion and compaction in the CNS. We suggest that loss of electrostatic cell-surface repulsion uncovers weak and unspecific attractive forces in the bilayer that bring the extracellular surfaces of a membrane into close contact over long distances.

Probing lipid membrane electrostatics

NASA Astrophysics Data System (ADS)

Yang, Yi

The electrostatic properties of lipid bilayer membranes play a significant role in many biological processes. Atomic force microscopy (AFM) is highly sensitive to membrane surface potential in electrolyte solutions. With fully characterized probe tips, AFM can perform quantitative electrostatic analysis of lipid membranes. Electrostatic interactions between Silicon nitride probes and supported zwitterionic dioleoylphosphatidylcholine (DOPC) bilayer with a variable fraction of anionic dioleoylphosphatidylserine (DOPS) were measured by AFM. Classical Gouy-Chapman theory was used to model the membrane electrostatics. The nonlinear Poisson-Boltzmann equation was numerically solved with finite element method to provide the potential distribution around the AFM tips. Theoretical tip-sample electrostatic interactions were calculated with the surface integral of both Maxwell and osmotic stress tensors on tip surface. The measured forces were interpreted with theoretical forces and the resulting surface charge densities of the membrane surfaces were in quantitative agreement with the Gouy-Chapman-Stern model of membrane charge regulation. It was demonstrated that the AFM can quantitatively detect membrane surface potential at a separation of several screening lengths, and that the AFM probe only perturbs the membrane surface potential by <2%. One important application of this technique is to estimate the dipole density of lipid membrane. Electrostatic analysis of DOPC lipid bilayers with the AFM reveals a repulsive force between the negatively charged probe tips and the zwitterionic lipid bilayers. This unexpected interaction has been analyzed quantitatively to reveal that the repulsion is due to a weak external field created by the internai membrane dipole moment. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported DOPC membranes. This new ability to quantitatively measure the membrane dipole density in a noninvasive manner will be useful in identifying the biological effects of the dipole potential. Finally, heterogeneous model membranes were studied with fluid electric force microscopy (FEFM). Electrostatic mapping was demonstrated with 50 nm resolution. The capabilities of quantitative electrostatic measurement and lateral charge density mapping make AFM a unique and powerful probe of membrane electrostatics.

Analysis of diffusion in curved surfaces and its application to tubular membranes.

PubMed

Klaus, Colin James Stockdale; Raghunathan, Krishnan; DiBenedetto, Emmanuele; Kenworthy, Anne K

2016-12-01

Diffusion of particles in curved surfaces is inherently complex compared with diffusion in a flat membrane, owing to the nonplanarity of the surface. The consequence of such nonplanar geometry on diffusion is poorly understood but is highly relevant in the case of cell membranes, which often adopt complex geometries. To address this question, we developed a new finite element approach to model diffusion on curved membrane surfaces based on solutions to Fick's law of diffusion and used this to study the effects of geometry on the entry of surface-bound particles into tubules by diffusion. We show that variations in tubule radius and length can distinctly alter diffusion gradients in tubules over biologically relevant timescales. In addition, we show that tubular structures tend to retain concentration gradients for a longer time compared with a comparable flat surface. These findings indicate that sorting of particles along the surfaces of tubules can arise simply as a geometric consequence of the curvature without any specific contribution from the membrane environment. Our studies provide a framework for modeling diffusion in curved surfaces and suggest that biological regulation can emerge purely from membrane geometry. © 2016 Klaus, Raghunathan, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Genetic dissection of early endosomal recycling highlights a TORC1-independent role for Rag GTPases

PubMed Central

2017-01-01

Endocytosed cell surface membrane proteins rely on recycling pathways for their return to the plasma membrane. Although endosome-to-plasma membrane recycling is critical for many cellular processes, much of the required machinery is unknown. We discovered that yeast has a recycling route from endosomes to the cell surface that functions efficiently after inactivation of the sec7-1 allele of Sec7, which controls transit through the Golgi. A genetic screen based on an engineered synthetic reporter that exclusively follows this pathway revealed that recycling was subject to metabolic control through the Rag GTPases Gtr1 and Gtr2, which work downstream of the exchange factor Vam6. Gtr1 and Gtr2 control the recycling pathway independently of TORC1 regulation through the Gtr1 interactor Ltv1. We further show that the early-endosome recycling route and its control though the Vam6>Gtr1/Gtr2>Ltv1 pathway plays a physiological role in regulating the abundance of amino acid transporters at the cell surface. PMID:28768685

Organelle-mimicking liposome dissociates G-quadruplexes and facilitates transcription

PubMed Central

Pramanik, Smritimoy; Tateishi-Karimata, Hisae; Sugimoto, Naoki

2014-01-01

Important biological reactions involving nucleic acids occur near the surface of membranes such as the nuclear membrane (NM) and rough endoplasmic reticulum (ER); however, the interactions between biomembranes and nucleic acids are poorly understood. We report here that transcription was facilitated in solution with liposomes, which mimic a biomembrane surface, relative to the reaction in a homogeneous aqueous solution when the template was able to form a G-quadruplex. The G-quadruplex is known to be an inhibitor of transcription, but the stability of the G-quadruplex was decreased at the liposome surface because of unfavourable enthalpy. The destabilization of the G-quadruplex was greater at the surface of NM- and ER-mimicking liposomes than at the surfaces of liposomes designed to mimic other organelles. Thermodynamic analyses revealed that the G-rich oligonucleotides adopted an extended structure at the liposome surface, whereas in solution the compact G-quadruplex was formed. Our data suggest that changes in structure and stability of nucleic acids regulate biological reactions at membrane surfaces. PMID:25336617

From the endoplasmic reticulum to the plasma membrane: mechanisms of CFTR folding and trafficking.

PubMed

Farinha, Carlos M; Canato, Sara

2017-01-01

CFTR biogenesis starts with its co-translational insertion into the membrane of endoplasmic reticulum and folding of the cytosolic domains, towards the acquisition of a fully folded compact native structure. Efficiency of this process is assessed by the ER quality control system that allows the exit of folded proteins but targets unfolded/misfolded CFTR to degradation. If allowed to leave the ER, CFTR is modified at the Golgi and reaches the post-Golgi compartments to be delivered to the plasma membrane where it functions as a cAMP- and phosphorylation-regulated chloride/bicarbonate channel. CFTR residence at the membrane is a balance of membrane delivery, endocytosis, and recycling. Several adaptors, motor, and scaffold proteins contribute to the regulation of CFTR stability and are involved in continuously assessing its structure through peripheral quality control systems. Regulation of CFTR biogenesis and traffic (and its dysregulation by mutations, such as the most common F508del) determine its overall activity and thus contribute to the fine modulation of chloride secretion and hydration of epithelial surfaces. This review covers old and recent knowledge on CFTR folding and trafficking from its synthesis to the regulation of its stability at the plasma membrane and highlights how several of these steps can be modulated to promote the rescue of mutant CFTR.

N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.

PubMed

Glozman, Rina; Okiyoneda, Tsukasa; Mulvihill, Cory M; Rini, James M; Barriere, Herve; Lukacs, Gergely L

2009-03-23

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

Passive flow regulators for drug delivery and hydrocephalus treatment

NASA Astrophysics Data System (ADS)

Chappel, E.; Dumont-Fillon, D.; Mefti, S.

2014-03-01

Passive flow regulators are usually intended to deliver or drain a fluid at a constant rate independently from pressure variations. New designs of passive flow regulators made of a stack of a silicon membrane anodically bonded to a Pyrex substrate are proposed. A first design has been built for the derivation of cerebrospinal fluid (CSF) towards peritoneum for hydrocephalus treatment. The device allows draining CSF at the patient production rate independently from postural changes. The flow rate is regulated at 20 ml/h in the range 10 to 40 mbar. Specific features to adjust in vivo the nominal flow rate are shown. A second design including high pressure shut-off feature has been made. The intended use is drug delivery with pressurized reservoir of typically 100 to 300 mbar. In both cases, the membrane comprises several holes facing pillars in the Pyrex substrate. These pillars are machined in a cavity which ensures a gap between the membrane and the pillars at rest. The fluid in the pressurized reservoir is directly in contact with the top surface of the membrane, inducing its deflection towards Pyrex substrate and closing progressively the fluidic pathway through each hole of the membrane. Since the membrane deflection is highly non-linear, FEM simulations have been performed to determine both radial position and diameter of the membrane holes that ensure a constant flow rate for a given range of pressure.

The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR

PubMed Central

Boncompain, Gaelle; Laketa, Vibor; Poser, Ina; Beck, Martin; Bork, Peer

2016-01-01

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane. PMID:27872256

Positive zeta potential of a negatively charged semi-permeable plasma membrane

NASA Astrophysics Data System (ADS)

Sinha, Shayandev; Jing, Haoyuan; Das, Siddhartha

2017-08-01

The negative charge of the plasma membrane (PM) severely affects the nature of moieties that may enter or leave the cells and controls a large number of ion-interaction-mediated intracellular and extracellular events. In this letter, we report our discovery of a most fascinating scenario, where one interface (e.g., membrane-cytosol interface) of the negatively charged PM shows a positive surface (or ζ) potential, while the other interface (e.g., membrane-electrolyte interface) still shows a negative ζ potential. Therefore, we encounter a completely unexpected situation where an interface (e.g., membrane-cytosol interface) that has a negative surface charge density demonstrates a positive ζ potential. We establish that the attainment of such a property by the membrane can be ascribed to an interplay of the nature of the membrane semi-permeability and the electrostatics of the electric double layer established on either side of the charged membrane. We anticipate that such a membrane property can lead to such capabilities of the cell (in terms of accepting or releasing certain kinds of moieties as well regulating cellular signaling) that was hitherto inconceivable.

Functional Characterization of LcpA, a Surface-Exposed Protein of Leptospira spp. That Binds the Human Complement Regulator C4BP▿

PubMed Central

Barbosa, Angela S.; Monaris, Denize; Silva, Ludmila B.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Cianciarullo, Aurora M.; Isaac, Lourdes; Abreu, Patricia A. E.

2010-01-01

We have previously shown that pathogenic leptospiral strains are able to bind C4b binding protein (C4BP). Surface-bound C4BP retains its cofactor activity, indicating that acquisition of this complement regulator may contribute to leptospiral serum resistance. In the present study, the abilities of seven recombinant putative leptospiral outer membrane proteins to interact with C4BP were evaluated. The protein encoded by LIC11947 interacted with this human complement regulator in a dose-dependent manner. The cofactor activity of C4BP bound to immobilized recombinant LIC11947 (rLIC11947) was confirmed by detecting factor I-mediated cleavage of C4b. rLIC11947 was therefore named LcpA (for leptospiral complement regulator-acquiring protein A). LcpA was shown to be an outer membrane protein by using immunoelectron microscopy, cell surface proteolysis, and Triton X-114 fractionation. The gene coding for LcpA is conserved among pathogenic leptospiral strains. This is the first characterization of a Leptospira surface protein that binds to the human complement regulator C4BP in a manner that allows this important regulator to control complement system activation mediated either by the classical pathway or by the lectin pathway. This newly identified protein may play a role in immune evasion by Leptospira spp. and may therefore represent a target for the development of a human vaccine against leptospirosis. PMID:20404075

WNK4 inhibits plasma membrane targeting of NCC through regulation of syntaxin13 SNARE formation.

PubMed

Chung, Woo Young; Park, Hyun Woo; Han, Jung Woo; Lee, Min Goo; Kim, Joo Young

2013-12-01

WNK4, a serine/threonine kinase, plays a critical role in the expression of membrane proteins in the cell surface; however, the underlying mechanism of WNK4 is not clear. Here, we demonstrate that WNK4 inhibits the fusion of plasma membrane delivering vesicle with sorting/recycling endosome through disrupting SNARE formation of syntaxin13, an endosomal t-SNARE and VAMP2, the v-SNARE in plasma membrane delivering vesicle. Their interaction and co-localization were enhanced by hyperosmotic stimulation which is known for WNK4 activation. The kinase domain of WNK4 interacts with the transmembrane domain (TM) of syntaxin13 and this interaction was abolished when the TM was replaced with that of syntaxin16. Interestingly, cell fractionation using sucrose gradients revealed that WNK4 inhibited the formation of the syntaxin13/VAMP2 SNARE complex in the endosomal compartment, but not syntaxin16/VAMP2 or syntaxin13/VAMP7. Syntaxin13 was not phosphorylated by WNK4 and WNK4KI also showed the same binding strength and similar inhibitory regulation on SNARE formation of syntaxin13. Physiological relevance of this mechanism was proved with the expression of NCC (Na(+) C1(-) co-transporter) in the cell surface. The inhibiting activity of WNK4 on surface expression of NCC was abolished by syntaxin13 siRNA transfection. These results suggest that WNK4 attenuates PM targeting of NCC proteins through regulation of syntaxin13 SNARE complex formation with VAMP2 in recycling and sorting endosome. © 2013.

Remodeling of the Host Cell Plasma Membrane by HIV-1 Nef and Vpu: A Strategy to Ensure Viral Fitness and Persistence.

PubMed

Sugden, Scott M; Bego, Mariana G; Pham, Tram N Q; Cohen, Éric A

2016-03-03

The plasma membrane protects the cell from its surroundings and regulates cellular communication, homing, and metabolism. Not surprisingly, the composition of this membrane is highly controlled through the vesicular trafficking of proteins to and from the cell surface. As intracellular pathogens, most viruses exploit the host plasma membrane to promote viral replication while avoiding immune detection. This is particularly true for the enveloped human immunodeficiency virus (HIV), which assembles and obtains its lipid shell directly at the plasma membrane. HIV-1 encodes two proteins, negative factor (Nef) and viral protein U (Vpu), which function primarily by altering the quantity and localization of cell surface molecules to increase virus fitness despite host antiviral immune responses. These proteins are expressed at different stages in the HIV-1 life cycle and employ a variety of mechanisms to target both unique and redundant surface proteins, including the viral receptor CD4, host restriction factors, immunoreceptors, homing molecules, tetraspanins and membrane transporters. In this review, we discuss recent progress in the study of the Nef and Vpu targeting of host membrane proteins with an emphasis on how remodeling of the cell membrane allows HIV-1 to avoid host antiviral immune responses leading to the establishment of systemic and persistent infection.

Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

PubMed Central

Goodwin, J. Shawn; Drake, Kimberly R.; Rogers, Carl; Wright, Latasha; Lippincott-Schwartz, Jennifer; Philips, Mark R.; Kenworthy, Anne K.

2005-01-01

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes. PMID:16027222

Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

PubMed Central

2015-01-01

The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

Hemocompatibility of poly(vinylidene fluoride) membrane grafted with network-like and brush-like antifouling layer controlled via plasma-induced surface PEGylation.

PubMed

Chang, Yung; Shih, Yu-Ju; Ko, Chao-Yin; Jhong, Jheng-Fong; Liu, Ying-Ling; Wei, Ta-Chin

2011-05-03

In this work, the hemocompatibility of PEGylated poly(vinylidene fluoride) (PVDF) microporous membranes with varying grafting coverage and structures via plasma-induced surface PEGylation was studied. Network-like and brush-like PEGylated layers on PVDF membrane surfaces were achieved by low-pressure and atmospheric plasma treatment. The chemical composition, physical morphology, grafting structure, surface hydrophilicity, and hydration capability of prepared membranes were determined to illustrate the correlations between grafting qualities and hemocompatibility of PEGylated PVDF membranes in contact with human blood. Plasma protein adsorption onto different PEGylated PVDF membranes from single-protein solutions and the complex medium of 100% human plasma were measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. Hemocompatibility of the PEGylated membranes was evaluated by the antifouling property of platelet adhesion observed by scanning electron microscopy (SEM) and the anticoagulant activity of the blood coagulant determined by testing plasma-clotting time. The control of grafting structures of PEGylated layers highly regulates the PVDF membrane to resist the adsorption of plasma proteins, the adhesion of platelets, and the coagulation of human plasma. It was found that PVDF membranes grafted with brush-like PEGylated layers presented higher hydration capability with binding water molecules than with network-like PEGylated layers to improve the hemocompatible character of plasma protein and blood platelet resistance in human blood. This work suggests that the hemocompatible nature of grafted PEGylated polymers by controlling grafting structures gives them great potential in the molecular design of antithrombogenic membranes for use in human blood.

Cell surface localization of the 78 kD glucose regulated protein (GRP 78) induced by thapsigargin.

PubMed

Delpino, A; Piselli, P; Vismara, D; Vendetti, S; Colizzi, V

1998-01-01

In the present study it was found that the synthesis of the 78 kD glucose-regulated protein (GRP 78 or BIP) is vigorously induced in human rabdomiosarcoma cells (TE 671/RD) following both short-term (1 h) and prolonged (18 h) exposure to 100 nM thapsigargin (Tg). Flow cytometric analysis with a specific anti-GRP 78 polyclonal antibody showed that Tg-treated cells express the GRP 78 on the plasma membrane. Cell surface localization of the Tg-induced GRP 78 was confirmed by biotinylation of membrane-exposed proteins and subsequent isolation of the biotin-labelled proteins by streptavidin/agarose affinity chromatography. It was found that a fraction of the Tg-induced GRP 78 is present among the biotin-labelled, surface-exposed, proteins. Conversely, the GRP 78 immunoprecipitated from unfractionated lysates of Tg-treated and biotin-reacted cells was found to be biotinylated. This is the first report demonstrating surface expression of GRP 78 in cells exposed to a specific GRP 78-inducing stimulus.

Membrane tension regulates clathrin-coated pit dynamics

NASA Astrophysics Data System (ADS)

Liu, Allen

2014-03-01

Intracellular organization depends on close communication between the extracellular environment and a network of cytoskeleton filaments. The interactions between cytoskeletal filaments and the plasma membrane lead to changes in membrane tension that in turns help regulate biological processes. Endocytosis is thought to be stimulated by low membrane tension and the removal of membrane increases membrane tension. While it is appreciated that the opposing effects of exocytosis and endocytosis have on keeping plasma membrane tension to a set point, it is not clear how membrane tension affects the dynamics of clathrin-coated pits (CCPs), the individual functional units of clathrin-mediated endocytosis. Furthermore, although it was recently shown that actin dynamics counteracts membrane tension during CCP formation, it is not clear what roles plasma membrane tension plays during CCP initiation. Based on the notion that plasma membrane tension is increased when the membrane area increases during cell spreading, we designed micro-patterned surfaces of different sizes to control the cell spreading sizes. Total internal reflection fluorescence microscopy of living cells and high content image analysis were used to quantify the dynamics of CCPs. We found that there is an increased proportion of CCPs with short (<20s) lifetime for cells on larger patterns. Interestingly, cells on larger patterns have higher CCP initiation density, an effect unexpected based on the conventional view of decreasing endocytosis with increasing membrane tension. Furthermore, by analyzing the intensity profiles of CCPs that were longer-lived, we found CCP intensity decreases with increasing cell size, indicating that the CCPs are smaller with increasing membrane tension. Finally, disruption of actin dynamics significantly increased the number of short-lived CCPs, but also decreased CCP initiation rate. Together, our study reveals new mechanistic insights into how plasma membrane tension regulates the dynamics of CCPs.

Trans-Membrane Area Asymmetry Controls the Shape of Cellular Organelles

PubMed Central

Beznoussenko, Galina V.; Pilyugin, Sergei S.; Geerts, Willie J. C.; Kozlov, Michael M.; Burger, Koert N. J.; Luini, Alberto; Derganc, Jure; Mironov, Alexander A.

2015-01-01

Membrane organelles often have complicated shapes and differ in their volume, surface area and membrane curvature. The ratio between the surface area of the cytosolic and luminal leaflets (trans-membrane area asymmetry (TAA)) determines the membrane curvature within different sites of the organelle. Thus, the shape of the organelle could be critically dependent on TAA. Here, using mathematical modeling and stereological measurements of TAA during fast transformation of organelle shapes, we present evidence that suggests that when organelle volume and surface area are constant, TAA can regulate transformation of the shape of the Golgi apparatus, endosomal multivesicular bodies, and microvilli of brush borders of kidney epithelial cells. Extraction of membrane curvature by small spheres, such as COPI-dependent vesicles within the Golgi (extraction of positive curvature), or by intraluminal vesicles within endosomes (extraction of negative curvature) controls the shape of these organelles. For instance, Golgi tubulation is critically dependent on the fusion of COPI vesicles with Golgi cisternae, and vice versa, for the extraction of membrane curvature into 50–60 nm vesicles, to induce transformation of Golgi tubules into cisternae. Also, formation of intraluminal ultra-small vesicles after fusion of endosomes allows equilibration of their TAA, volume and surface area. Finally, when microvilli of the brush border are broken into vesicles and microvilli fragments, TAA of these membranes remains the same as TAA of the microvilli. Thus, TAA has a significant role in transformation of organelle shape when other factors remain constant. PMID:25761238

Versatile and Rapid Postfunctionalization from Cyclodextrin Modified Host Polymeric Membrane Substrate.

PubMed

Deng, Jie; Liu, Xinyue; Zhang, Shuqing; Cheng, Chong; Nie, Chuanxiong; Zhao, Changsheng

2015-09-08

Surface modification has long been of great interest to impart desired functionalities to the bioimplants. However, due to the limitations of recent technologies in surface modification, it is highly desirable to explore novel protocols, which can advantageously and efficiently endow the inert material surfaces with versatile biofunctionalities. Herein, to achieve versatile and rapid postfunctionalization of polymeric membrane, we demonstrate a new strategy for the fabrication of β-cyclodextrin (β-CD) modified host membrane substrate that can recognize a series of well-designed guest macromolecules. The surface assembly procedure was driven by the host-guest interaction between adamantane (Ad) and β-CD. β-CD immobilized host membrane was fabricated via two steps: (1) epoxy groups enriched poly(ether sulfone) (PES) membrane was first prepared via in situ cross-linking polymerization and subsequently phase separation; (2) mono-6-deoxy-6-ethylenediamine-β-CD (EDA-β-CD) was then anchored onto the surface of the epoxy functionalized PES membrane to obtain PES-CD. Subsequently, three types of Ad-terminated polymers, including Ad-poly(styrenesulfonate-co-sodium acrylate) (Ad-PSA), Ad-methoxypoly(ethylene glycol) (Ad-PEG), and Ad-poly(methyl chloride-quaternized 2-(dimethylamino)ethyl methacrylate (Ad-PMT), were separately assembled onto the β-CD immobilized surfaces to endow the membranes with anticoagulant, antifouling, and antibacterial capability, respectively. Activated partial thromboplastin time (APTT), thrombin time (TT), and prothrombin time (PT) measurements were carried out to explore the anticoagulant activity. The antifouling capability was evaluated via protein adsorption and platelet adhesion measurements. Moreover, Staphyllococcous aureus (S. aureus) was selected as model bacteria to evaluate the antibacterial ability of the functionalized membranes. The results indicated that well-regulated blood compatibility, antifouling capability, and bactericidal activity could be achieved by the proposed rapid postfunctionalization on polymeric membranes. This approach of versatile and rapid postfunctionalization is promising for the preparation of multifunctional polymeric membrane materials to meet with various demands for the further applications.

Imaging of blood plasma coagulation at supported lipid membranes.

PubMed

Faxälv, Lars; Hume, Jasmin; Kasemo, Bengt; Svedhem, Sofia

2011-12-15

The blood coagulation system relies on lipid membrane constituents to act as regulators of the coagulation process upon vascular trauma, and in particular the 2D configuration of the lipid membranes is known to efficiently catalyze enzymatic activity of blood coagulation factors. This work demonstrates a new application of a recently developed methodology to study blood coagulation at lipid membrane interfaces with the use of imaging technology. Lipid membranes with varied net charges were formed on silica supports by systematically using different combinations of lipids where neutral phosphocholine (PC) lipids were mixed with phospholipids having either positively charged ethylphosphocholine (EPC), or negatively charged phosphatidylserine (PS) headgroups. Coagulation imaging demonstrated that negatively charged SiO(2) and membrane surfaces exposing PS (obtained from liposomes containing 30% of PS) had coagulation times which were significantly shorter than those for plain PC membranes and EPC exposing membrane surfaces (obtained from liposomes containing 30% of EPC). Coagulation times decreased non-linearly with increasing negative surface charge for lipid membranes. A threshold value for shorter coagulation times was observed below a PS content of ∼6%. We conclude that the lipid membranes on solid support studied with the imaging setup as presented in this study offers a flexible and non-expensive solution for coagulation studies at biological membranes. It will be interesting to extend the present study towards examining coagulation on more complex lipid-based model systems. Copyright © 2011 Elsevier Inc. All rights reserved.

Role of ER Export Signals in Controlling Surface Potassium Channel Numbers

NASA Astrophysics Data System (ADS)

Ma, Dzwokai; Zerangue, Noa; Lin, Yu-Fung; Collins, Anthony; Yu, Mei; Jan, Yuh Nung; Yeh Jan, Lily

2001-01-01

Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.

Unconventional secretory processing diversifies neuronal ion channel properties

PubMed Central

Hanus, Cyril; Geptin, Helene; Tushev, Georgi; Garg, Sakshi; Alvarez-Castelao, Beatriz; Sambandan, Sivakumar; Kochen, Lisa; Hafner, Anne-Sophie; Langer, Julian D; Schuman, Erin M

2016-01-01

N-glycosylation – the sequential addition of complex sugars to adhesion proteins, neurotransmitter receptors, ion channels and secreted trophic factors as they progress through the endoplasmic reticulum and the Golgi apparatus – is one of the most frequent protein modifications. In mammals, most organ-specific N-glycosylation events occur in the brain. Yet, little is known about the nature, function and regulation of N-glycosylation in neurons. Using imaging, quantitative immunoblotting and mass spectrometry, we show that hundreds of neuronal surface membrane proteins are core-glycosylated, resulting in the neuronal membrane displaying surprisingly high levels of glycosylation profiles that are classically associated with immature intracellular proteins. We report that while N-glycosylation is generally required for dendritic development and glutamate receptor surface expression, core-glycosylated proteins are sufficient to sustain these processes, and are thus functional. This atypical glycosylation of surface neuronal proteins can be attributed to a bypass or a hypo-function of the Golgi apparatus. Core-glycosylation is regulated by synaptic activity, modulates synaptic signaling and accelerates the turnover of GluA2-containing glutamate receptors, revealing a novel mechanism that controls the composition and sensing properties of the neuronal membrane. DOI: http://dx.doi.org/10.7554/eLife.20609.001 PMID:27677849

ADP-Ribosylation Factor 6 Regulates a Novel Plasma Membrane Recycling Pathway

PubMed Central

Radhakrishna, Harish; Donaldson, Julie G.

1997-01-01

ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor α subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton. PMID:9314528

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

PubMed

Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

2014-10-13

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

PubMed Central

Caiolfa, Valeria R.; Zamai, Moreno; Malengo, Gabriele; Andolfo, Annapaola; Madsen, Chris D.; Sutin, Jason; Digman, Michelle A.; Gratton, Enrico; Blasi, Francesco; Sidenius, Nicolai

2007-01-01

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. PMID:18056417

Cloning of B cell-specific membrane tetraspanning molecule BTS possessing B cell proliferation-inhibitory function.

PubMed

Suenaga, Tadahiro; Arase, Hisashi; Yamasaki, Sho; Kohno, Masayuki; Yokosuka, Tadashi; Takeuchi, Arata; Hattori, Takamichi; Saito, Takashi

2007-11-01

Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.

Nanoporous membranes with electrochemically switchable, chemically stabilized ionic selectivity

NASA Astrophysics Data System (ADS)

Small, Leo J.; Wheeler, David R.; Spoerke, Erik D.

2015-10-01

Nanopore size, shape, and surface charge all play important roles in regulating ionic transport through nanoporous membranes. The ability to control these parameters in situ provides a means to create ion transport systems tunable in real time. Here, we present a new strategy to address this challenge, utilizing three unique electrochemically switchable chemistries to manipulate the terminal functional group and control the resulting surface charge throughout ensembles of gold plated nanopores in ion-tracked polycarbonate membranes 3 cm2 in area. We demonstrate the diazonium mediated surface functionalization with (1) nitrophenyl chemistry, (2) quinone chemistry, and (3) previously unreported trimethyl lock chemistry. Unlike other works, these chemistries are chemically stabilized, eliminating the need for a continuously applied gate voltage to maintain a given state and retain ionic selectivity. The effect of surface functionalization and nanopore geometry on selective ion transport through these functionalized membranes is characterized in aqueous solutions of sodium chloride at pH = 5.7. The nitrophenyl surface allows for ionic selectivity to be irreversibly switched in situ from cation-selective to anion-selective upon reduction to an aminophenyl surface. The quinone-terminated surface enables reversible changes between no ionic selectivity and a slight cationic selectivity. Alternatively, the trimethyl lock allows ionic selectivity to be reversibly switched by up to a factor of 8, approaching ideal selectivity, as a carboxylic acid group is electrochemically revealed or hidden. By varying the pore shape from cylindrical to conical, it is demonstrated that a controllable directionality can be imparted to the ionic selectivity. Combining control of nanopore geometry with stable, switchable chemistries facilitates superior control of molecular transport across the membrane, enabling tunable ion transport systems.Nanopore size, shape, and surface charge all play important roles in regulating ionic transport through nanoporous membranes. The ability to control these parameters in situ provides a means to create ion transport systems tunable in real time. Here, we present a new strategy to address this challenge, utilizing three unique electrochemically switchable chemistries to manipulate the terminal functional group and control the resulting surface charge throughout ensembles of gold plated nanopores in ion-tracked polycarbonate membranes 3 cm2 in area. We demonstrate the diazonium mediated surface functionalization with (1) nitrophenyl chemistry, (2) quinone chemistry, and (3) previously unreported trimethyl lock chemistry. Unlike other works, these chemistries are chemically stabilized, eliminating the need for a continuously applied gate voltage to maintain a given state and retain ionic selectivity. The effect of surface functionalization and nanopore geometry on selective ion transport through these functionalized membranes is characterized in aqueous solutions of sodium chloride at pH = 5.7. The nitrophenyl surface allows for ionic selectivity to be irreversibly switched in situ from cation-selective to anion-selective upon reduction to an aminophenyl surface. The quinone-terminated surface enables reversible changes between no ionic selectivity and a slight cationic selectivity. Alternatively, the trimethyl lock allows ionic selectivity to be reversibly switched by up to a factor of 8, approaching ideal selectivity, as a carboxylic acid group is electrochemically revealed or hidden. By varying the pore shape from cylindrical to conical, it is demonstrated that a controllable directionality can be imparted to the ionic selectivity. Combining control of nanopore geometry with stable, switchable chemistries facilitates superior control of molecular transport across the membrane, enabling tunable ion transport systems. Electronic supplementary information (ESI) available: Experimental procedures, synthesis, and characterization of molecules 1, 2 and 3. Explanation of the electrochemical method for approximating nanopore diameter. Additional XPS spectra. See DOI: 10.1039/C5NR02939B

Rapid Steroid Hormone Actions Initiated at the Cell Surface and the Receptors that Mediate Them with an Emphasis on Recent Progress in Fish Models

PubMed Central

Thomas, Peter

2011-01-01

In addition to the classic genomic mechanism of steroid action mediated by activation of intracellular nuclear receptors, there is now extensive evidence that steroids also activate receptors on the cell surface to initiate rapid intracellular signaling and biological responses that are often nongenomic. Recent progress in our understanding of rapid, cell surface-initiated actions of estrogens, progestins, androgens and corticosteroids and the identities of the membrane receptors that act as their intermediaries is briefly reviewed with a special emphasis on studies in teleost fish. Two recently discovered novel proteins with seven-transmembrane domains, G protein-coupled receptor 30 (GPR30), and membrane progestin receptors (mPRs) have the ligand binding and signaling characteristics of estrogen and progestin membrane receptors, respectively, but their functional significance is disputed by some researchers. GPR30 is expressed on the cell surface of fish oocytes and mediates estrogen inhibition of oocyte maturation. mPRα is also expressed on the oocyte cell surface and is the intermediary in progestin induction of oocyte maturation in fish. Recent results suggest there is cross-talk between these two hormonal pathways and that there is reciprocal down-regulation of GPR30 and mPRα expression by estrogens and progestins at different phases of oocyte development to regulate the onset of oocyte maturation. There is also evidence in fish that mPRs are involved in progestin induction of sperm hypermotility and anti-apoptotic actions in ovarian follicle cells. Nonclassical androgen and corticosteroid actions have also been described in fish models but the membrane receptors mediating these actions have not been identified. PMID:22154643

Understanding the Capsanthin Tails in Regulating the Hydrophilic-Lipophilic Balance of Carbon Dots for a Rapid Crossing Cell Membrane.

PubMed

Chen, Jing; Zhang, Xiang; Zhang, Ye; Wang, Wei; Li, Shuya; Wang, Yucai; Hu, Mengyue; Liu, Li; Bi, Hong

2017-10-03

Here we use natural Chinese paprika to prepare a new kind of amphiphilic carbon dot (A-Dot) that exhibits bright, multicolored fluorescence and contains hydrophilic groups as well as lipophilic capsanthin tails on the surface. It is found that the capsanthin tails in a phospholipid-like structure can promote cell internalization of the A-Dots via crossing cell membranes rapidly in an energy-independent fashion. Compared to highly hydrophilic carbon dots (H-Dots), a control sample prepared from the microwave thermolysis of citric acid and ethylenediamine, our synthesized A-Dots can be taken up by CHO, HeLa, and HFF cells more easily. More importantly, we develop a method to calibrate the hydrophilic-lipophilic balance (HLB) values of various kinds of carbon dots (C-Dots). HLB values of A-Dots and H-Dots are determined to be 6.4 and 18.4, respectively. Moreover, we discover that the cellular uptake efficiency of C-Dots is closely related to their HLBs, and the C-Dots with an HLB value of around 6.4 cross the cell membrane easier and faster. As we regulate the HLB value of the A-Dots from 6.4 to 15.3 by removing the capsanthin tails from their surfaces via alkali refluxing, it is found that the refluxed A-Dots can hardly cross HeLa cell membranes. Our work is an essential step toward understanding the importance of regulating the HLB values as well as the surface polarity of the C-Dots for their practical use in bioimaging and also provides a simple but effective way to judge whether the C-Dots in hand are appropriate for cell imaging.

The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

PubMed

Steenblock, Charlotte; Heckel, Tobias; Czupalla, Cornelia; Espírito Santo, Ana Isabel; Niehage, Christian; Sztacho, Martin; Hoflack, Bernard

2014-06-27

The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Capacitance measurements of regulated exocytosis in mouse taste cells.

PubMed

Vandenbeuch, Aurelie; Zorec, Robert; Kinnamon, Sue C

2010-11-03

Exocytosis, consisting of the merger of vesicle and plasma membrane, is a common mechanism used by different types of nucleated cells to release their vesicular contents. Taste cells possess vesicles containing various neurotransmitters to communicate with adjacent taste cells and afferent nerve fibers. However, whether these vesicles engage in exocytosis on a stimulus is not known. Since vesicle membrane merger with the plasma membrane is reflected in plasma membrane area fluctuations, we measured membrane capacitance (C(m)), a parameter linearly related to membrane surface area. To investigate whether taste cells undergo regulated exocytosis, we used the compensated tight-seal whole-cell recording technique to monitor depolarization-induced changes in C(m) in the different types of taste cells. To identify taste cell types, mice expressing green fluorescent protein from the TRPM5 promoter or from the GAD67 promoter were used to discriminate type II and type III taste cells, respectively. Moreover, the cell types were also identified by monitoring their voltage-current properties. The results demonstrate that only type III taste cells show significant depolarization-induced increases in C(m), which were correlated to the voltage-activated calcium currents. The results suggest that type III, but neither type II nor type I cells exhibit depolarization-induced regulated exocytosis to release transmitter and activate gustatory afferent nerve fibers.

REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

PubMed Central

Ho, Vincent K.; Angelotti, Timothy

2013-01-01

Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins. Therefore, some REEPs can be further described as ER membrane shaping adapter proteins. PMID:24098485

Cholesterol Modulates CFTR Confinement in the Plasma Membrane of Primary Epithelial Cells

PubMed Central

Abu-Arish, Asmahan; Pandzic, Elvis; Goepp, Julie; Matthes, Elizabeth; Hanrahan, John W.; Wiseman, Paul W.

2015-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function. PMID:26153705

Coupled Segmentation of Nuclear and Membrane-bound Macromolecules through Voting and Multiphase Level Set

PubMed Central

Wen, Quan

2014-01-01

Membrane-bound macromolecules play an important role in tissue architecture and cell-cell communication, and is regulated by almost one-third of the genome. At the optical scale, one group of membrane proteins expresses themselves as linear structures along the cell surface boundaries, while others are sequestered; and this paper targets the former group. Segmentation of these membrane proteins on a cell-by-cell basis enables the quantitative assessment of localization for comparative analysis. However, such membrane proteins typically lack continuity, and their intensity distributions are often very heterogeneous; moreover, nuclei can form large clump, which further impedes the quantification of membrane signals on a cell-by-cell basis. To tackle these problems, we introduce a three-step process to (i) regularize the membrane signal through iterative tangential voting, (ii) constrain the location of surface proteins by nuclear features, where clumps of nuclei are segmented through a delaunay triangulation approach, and (iii) assign membrane-bound macromolecules to individual cells through an application of multi-phase geodesic level-set. We have validated our method using both synthetic data and a dataset of 200 images, and are able to demonstrate the efficacy of our approach with superior performance. PMID:25530633

Specialized sorting of GLUT4 and its recruitment to the cell surface are independently regulated by distinct Rabs

PubMed Central

Sadacca, L. Amanda; Bruno, Joanne; Wen, Jennifer; Xiong, Wenyong; McGraw, Timothy E.

2013-01-01

Adipocyte glucose uptake in response to insulin is essential for physiological glucose homeostasis: stimulation of adipocytes with insulin results in insertion of the glucose transporter GLUT4 into the plasma membrane and subsequent glucose uptake. Here we establish that RAB10 and RAB14 are key regulators of GLUT4 trafficking that function at independent, sequential steps of GLUT4 translocation. RAB14 functions upstream of RAB10 in the sorting of GLUT4 to the specialized transport vesicles that ferry GLUT4 to the plasma membrane. RAB10 and its GTPase-activating protein (GAP) AS160 comprise the principal signaling module downstream of insulin receptor activation that regulates the accumulation of GLUT4 transport vesicles at the plasma membrane. Although both RAB10 and RAB14 are regulated by the GAP activity of AS160 in vitro, only RAB10 is under the control of AS160 in vivo. Insulin regulation of the pool of RAB10 required for GLUT4 translocation occurs through regulation of AS160, since activation of RAB10 by DENND4C, its GTP exchange factor, does not require insulin stimulation. PMID:23804653

Elucidating the Functional Roles of Spatial Organization in Cross-Membrane Signal Transduction by a Hybrid Simulation Method.

PubMed

Chen, Jiawen; Xie, Zhong-Ru; Wu, Yinghao

2016-07-01

The ligand-binding of membrane receptors on cell surfaces initiates the dynamic process of cross-membrane signal transduction. It is an indispensable part of the signaling network for cells to communicate with external environments. Recent experiments revealed that molecular components in signal transduction are not randomly mixed, but spatially organized into distinctive patterns. These patterns, such as receptor clustering and ligand oligomerization, lead to very different gene expression profiles. However, little is understood about the molecular mechanisms and functional impacts of this spatial-temporal regulation in cross-membrane signal transduction. In order to tackle this problem, we developed a hybrid computational method that decomposes a model of signaling network into two simulation modules. The physical process of binding between receptors and ligands on cell surfaces are simulated by a diffusion-reaction algorithm, while the downstream biochemical reactions are modeled by stochastic simulation of Gillespie algorithm. These two processes are coupled together by a synchronization framework. Using this method, we tested the dynamics of a simple signaling network in which the ligand binding of cell surface receptors triggers the phosphorylation of protein kinases, and in turn regulates the expression of target genes. We found that spatial aggregation of membrane receptors at cellular interfaces is able to either amplify or inhibit downstream signaling outputs, depending on the details of clustering mechanism. Moreover, by providing higher binding avidity, the co-localization of ligands into multi-valence complex modulates signaling in very different ways that are closely related to the binding affinity between ligand and receptor. We also found that the temporal oscillation of the signaling pathway that is derived from genetic feedback loops can be modified by the spatial clustering of membrane receptors. In summary, our method demonstrates the functional importance of spatial organization in cross-membrane signal transduction. The method can be applied to any specific signaling pathway in cells.

CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface

PubMed Central

Achour, Lamia; Scott, Mark G.H.; Shirvani, Hamasseh; Thuret, Alain; Bismuth, Georges; Labbé-Jullié, Catherine; Marullo, Stefano

2009-01-01

The association of CD4, a glycoprotein involved in T cell development and antigen recognition, and CCR5, a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for the human immunodeficiency virus HIV. We observed that the vast majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relative low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration–dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cell distribution of CXCR4, the other HIV co-receptor. These results reveal a previously unappreciated role of CD4, which contributes to regulate CCR5 export to the plasma membrane. PMID:19064722

Calcium-activated potassium channels in basolateral membranes of colon epithelial cells; reconstitution and functional properties.

PubMed

Wiener, H; Turnheim, K

1990-10-26

Using differential sedimentation, isopycnic and Ficoll-400 barrier centrifugation, basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon were enriched 34- and 9-fold, respectively. 86Rb(+)-uptake into these vesicles, driven by an electrical potential difference, was stimulated by submicromolar Ca2+ activities and inhibited by Ba2+. These findings indicate the presence of Ca2(+)-activated K+ channels. The K+ channels in surface and crypt cell membranes differed with respect to inhibition by the bee venom apamin, the scorpion venom charybdotoxin and tetraethylammonium and exhibited a different pH dependence. Fusion of basolateral membrane vesicles with planar phospholipid bilayers revealed the presence of high-conductance Ba2(+)-sensitive K+ channels which were activated by micromolar Ca2+ and inhibited by crude scorpion venom and trifluoperazine. These K+ channels may be involved in the coupling of apical and basolateral membrane conductances during Na+ absorption and Cl- secretion, but they may also play a role in cell volume regulation.

Cargo-mediated regulation of a rapid Rab4-dependent recycling pathway.

PubMed

Yudowski, Guillermo A; Puthenveedu, Manojkumar A; Henry, Anastasia G; von Zastrow, Mark

2009-06-01

Membrane trafficking is well known to regulate receptor-mediated signaling processes, but less is known about whether signaling receptors conversely regulate the membrane trafficking machinery. We investigated this question by focusing on the beta-2 adrenergic receptor (B2AR), a G protein-coupled receptor whose cellular signaling activity is controlled by ligand-induced endocytosis followed by recycling. We used total internal reflection fluorescence microscopy (TIR-FM) and tagging with a pH-sensitive GFP variant to image discrete membrane trafficking events mediating B2AR endo- and exocytosis. Within several minutes after initiating rapid endocytosis of B2ARs by the adrenergic agonist isoproterenol, we observed bright "puffs" of locally increased surface fluorescence intensity representing discrete Rab4-dependent recycling events. These events reached a constant frequency in the continuous presence of isoproterenol, and agonist removal produced a rapid (observed within 1 min) and pronounced (approximately twofold) increase in recycling event frequency. This regulation required receptor signaling via the cAMP-dependent protein kinase (PKA) and a specific PKA consensus site located in the carboxyl-terminal cytoplasmic tail of the B2AR itself. B2AR-mediated regulation was not restricted to this membrane cargo, however, as transferrin receptors packaged in the same population of recycling vesicles were similarly affected. In contrast, net recycling measured over a longer time interval (10 to 30 min) was not detectably regulated by B2AR signaling. These results identify rapid regulation of a specific recycling pathway by a signaling receptor cargo.

A Stable Human-Cell System Overexpressing Cystic Fibrosis Transmembrane Conductance Regulator Recombinant Protein at the Cell Surface

PubMed Central

Dai, Qun; Aleksandrov, Andrei A.; Bajrami, Bekim; Diego, Pamela Ann; Wu, Xing; Ray, Marjorie; Naren, Anjaparavanda P.; Riordan, John R.; Yao, Xudong; DeLucas, Lawrence J.; Urbatsch, Ina L.; Kappes, John C.

2015-01-01

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, or an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTRFLAG-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTRFLAG-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment futrure CF drug discovery efforts, including biophysical and structural studies. PMID:25577540

The membrane trafficking and functionality of the K+-Cl- co-transporter KCC2 is regulated by TGF-β2.

PubMed

Roussa, Eleni; Speer, Jan Manuel; Chudotvorova, Ilona; Khakipoor, Shokoufeh; Smirnov, Sergei; Rivera, Claudio; Krieglstein, Kerstin

2016-09-15

Functional activation of the neuronal K(+)-Cl(-) co-transporter KCC2 (also known as SLC12A5) is a prerequisite for shifting GABAA responses from depolarizing to hyperpolarizing during development. Here, we introduce transforming growth factor β2 (TGF-β2) as a new regulator of KCC2 membrane trafficking and functional activation. TGF-β2 controls membrane trafficking, surface expression and activity of KCC2 in developing and mature mouse primary hippocampal neurons, as determined by immunoblotting, immunofluorescence, biotinylation of surface proteins and KCC2-mediated Cl(-) extrusion. We also identify the signaling pathway from TGF-β2 to cAMP-response-element-binding protein (CREB) and Ras-associated binding protein 11b (Rab11b) as the underlying mechanism for TGF-β2-mediated KCC2 trafficking and functional activation. TGF-β2 increases colocalization and interaction of KCC2 with Rab11b, as determined by 3D stimulated emission depletion (STED) microscopy and co-immunoprecipitation, respectively, induces CREB phosphorylation, and enhances Rab11b gene expression. Loss of function of either CREB1 or Rab11b suppressed TGF-β2-dependent KCC2 trafficking, surface expression and functionality. Thus, TGF-β2 is a new regulatory factor for KCC2 functional activation and membrane trafficking, and a putative indispensable molecular determinant for the developmental shift of GABAergic transmission. © 2016. Published by The Company of Biologists Ltd.

Differential regulation of membrane-associated mucins in the human ocular surface epithelium.

PubMed

Hori, Yuichi; Spurr-Michaud, Sandra; Russo, Cindy Leigh; Argüeso, Pablo; Gipson, Ilene K

2004-01-01

Membrane-associated mucins present in the apical cells of the ocular surface epithelium (MUC1, -4, and -16) are believed to contribute to the maintenance of a hydrated and wet-surfaced epithelial phenotype. Serum and retinoic acid (RA) have been used to treat drying ocular surface diseases. The goal of this study was to determine whether serum or RA regulates the production of membrane-associated mucins in human conjunctival epithelial cells. A telomerase-immortalized human conjunctival epithelial cell line (HCjE) was used. Cells were cultured in serum-free medium to confluence and then cultured with either 10% calf serum or with 100 nM RA for 0 to 72 hours. Conventional RT-PCR was used to determine the expression of retinoic acid receptors (RARs) and quantitative real-time PCR was used to investigate the mRNA expression of MUC1, -4, and -16. Protein levels were assayed by immunoblot analysis, using the antibodies HMFG-2, 1G8, or OC125, which are specific to MUC1, -4 and -16, respectively. To determine whether RA-associated MUC4 mRNA induction is a direct or indirect effect, HCjE cells were treated with RA and the protein synthesis inhibitor cycloheximide (1.0 microg/mL) for 12 hours. MUC1 and -16, but not -4, mRNAs were detectable in HCjE cells grown in serum-free medium. Real-time PCR revealed that MUC4 mRNA was significantly induced by serum 3 hours after its addition, and that MUC1 and MUC16 mRNA levels were significantly upregulated at 72 hours. Western blot analysis demonstrated that the MUC1, -4, and -16 proteins increased over time after addition of serum. Conventional RT-PCR analysis demonstrated that RAR-alpha and -gamma mRNA were expressed in native human conjunctival tissue as well as in the HCjE cells. Treatment with RA upregulated the expression of both MUC4 and -16 mRNA and protein, but MUC1 was unaffected. Because the protein synthesis inhibitor cycloheximide did not prevent the RA-associated induction of MUC4 mRNA, the action of RA on the MUC4 promoter may be direct. The membrane-associated mucins of the ocular surface epithelia, MUC1, -4, and -16, are differentially regulated by serum and RA in the telomerase-immortalized human conjunctival epithelial cell line. Serum derived from vessels in the conjunctiva may play an important role in mucin regulation in the ocular surface epithelia. These data also support the clinical efficacy of autologous serum and RA application in patients with ocular surface diseases. Furthermore, the data suggest that MUC4 and -16 are particularly important hydrophilic molecules involved in maintenance of a healthy ocular surface.

[Membrane model of the regulation of proliferation: the theory and interpretation of an experiment].

PubMed

Volkov, E I

1983-04-01

The role of cell surface physical organization in the cell cycle regulation is analyzed within the framework of the earlier proposed theory (Chernavskii et al., 1982). Two models of cell surface are considered: hard-frame fluid-mosaic model (latticemosaic) and the fluid-mosaic one. The former deals with normal cells. The existence of integral carcasse or "frame" which is formed by the essential part of cross-linked membrane components and may have at least two different conformational states is hypothesized. The second model describes membranes of tumour cells. With the latter theory any mitogen (excluding the restoration of nutrient depletion) reduces the mechanical tensile strength of the frame and stimulates the general structural rearrangement of the plasma membrane. There are only two conformational transitions during the cell cycle which serve as signals for the beginning of S and M phases. If the values of tensile strength are great enough and therefore the conformational transitions are impossible, the cells pass into the resting (prereplicative--G01, or premitotical--G02) state. Three types of experiments are interpreted in the proposed theory: a) on differences in the action of growth factors on normal and tumour cell cycle, b) on the necessary condition for mitogenicity of lectins, c) on the stimulation of proliferation by mechanical deformation of cells.

Computational and theoretical approaches for studies of a lipid recognition protein on biological membranes

PubMed Central

Yamamoto, Eiji

2017-01-01

Many cellular functions, including cell signaling and related events, are regulated by the association of peripheral membrane proteins (PMPs) with biological membranes containing anionic lipids, e.g., phosphatidylinositol phosphate (PIP). This association is often mediated by lipid recognition modules present in many PMPs. Here, I summarize computational and theoretical approaches to investigate the molecular details of the interactions and dynamics of a lipid recognition module, the pleckstrin homology (PH) domain, on biological membranes. Multiscale molecular dynamics simulations using combinations of atomistic and coarse-grained models yielded results comparable to those of actual experiments and could be used to elucidate the molecular mechanisms of the formation of protein/lipid complexes on membrane surfaces, which are often difficult to obtain using experimental techniques. Simulations revealed some modes of membrane localization and interactions of PH domains with membranes in addition to the canonical binding mode. In the last part of this review, I address the dynamics of PH domains on the membrane surface. Local PIP clusters formed around the proteins exhibit anomalous fluctuations. This dynamic change in protein-lipid interactions cause temporally fluctuating diffusivity of proteins, i.e., the short-term diffusivity of the bound protein changes substantially with time, and may in turn contribute to the formation/dissolution of protein complexes in membranes. PMID:29159013

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

PubMed Central

Farr, Glen A.; Hull, Michael; Mellman, Ira

2009-01-01

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells. PMID:19620635

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

PubMed

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

2015-03-01

Cell-cell fusion between gametes is a defining step during development of eukaryotes, yet we know little about the cellular and molecular mechanisms of the gamete membrane fusion reaction. HAP2 is the sole gamete-specific protein in any system that is broadly conserved and shown by gene disruption to be essential for gamete fusion. The wide evolutionary distribution of HAP2 (also known as GCS1) indicates it was present in the last eukaryotic common ancestor and, therefore, dissecting its molecular properties should provide new insights into fundamental features of fertilization. HAP2 acts at a step after membrane adhesion, presumably directly in the merger of the lipid bilayers. Here, we use the unicellular alga Chlamydomonas to characterize contributions of key regions of HAP2 to protein location and function. We report that mutation of three strongly conserved residues in the ectodomain has no effect on targeting or fusion, although short deletions that include those residues block surface expression and fusion. Furthermore, HAP2 lacking a 237-residue segment of the cytoplasmic region is expressed at the cell surface, but fails to localize at the apical membrane patch specialized for fusion and fails to rescue fusion. Finally, we provide evidence that the ancient HAP2 contained a juxta-membrane, multi-cysteine motif in its cytoplasmic region, and that mutation of a cysteine dyad in this motif preserves protein localization, but substantially impairs HAP2 fusion activity. Thus, the ectodomain of HAP2 is essential for its surface expression, and the cytoplasmic region targets HAP2 to the site of fusion and regulates the fusion reaction. © 2015. Published by The Company of Biologists Ltd.

Hyaluronan, CD44, and Emmprin Regulate Lactate Efflux and Membrane Localization of Monocarboxylate Transporters in Human Breast Carcinoma Cells

PubMed Central

Slomiany, Mark G.; Grass, G. Daniel; Robertson, Angela D.; Yang, Xiao Y.; Maria, Bernard L.; Beeson, Craig; Toole, Bryan P.

2013-01-01

Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin)is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells. PMID:19176383

The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins.

PubMed

Kawada, Daiki; Kobayashi, Hiromu; Tomita, Tsuyoshi; Nakata, Eisuke; Nagano, Makoto; Siekhaus, Daria Elisabeth; Toshima, Junko Y; Toshima, Jiro

2015-01-01

Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Cholesterol modulates CFTR confinement in the plasma membrane of primary epithelial cells.

PubMed

Abu-Arish, Asmahan; Pandzic, Elvis; Goepp, Julie; Matthes, Elizabeth; Hanrahan, John W; Wiseman, Paul W

2015-07-07

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma-membrane anion channel that, when mutated, causes the disease cystic fibrosis. Although CFTR has been detected in a detergent-resistant membrane fraction prepared from airway epithelial cells, suggesting that it may partition into cholesterol-rich membrane microdomains (lipid rafts), its compartmentalization has not been demonstrated in intact cells and the influence of microdomains on CFTR lateral mobility is unknown. We used live-cell imaging, spatial image correlation spectroscopy, and k-space image correlation spectroscopy to examine the aggregation state of CFTR and its dynamics both within and outside microdomains in the plasma membrane of primary human bronchial epithelial cells. These studies were also performed during treatments that augment or deplete membrane cholesterol. We found two populations of CFTR molecules that were distinguishable based on their dynamics at the cell surface. One population showed confinement and had slow dynamics that were highly cholesterol dependent. The other, more abundant population was less confined and diffused more rapidly. Treatments that deplete the membrane of cholesterol caused the confined fraction and average number of CFTR molecules per cluster to decrease. Elevating cholesterol had the opposite effect, increasing channel aggregation and the fraction of channels displaying confinement, consistent with CFTR recruitment into cholesterol-rich microdomains with dimensions below the optical resolution limit. Viral infection caused the nanoscale microdomains to fuse into large platforms and reduced CFTR mobility. To our knowledge, these results provide the first biophysical evidence for multiple CFTR populations and have implications for regulation of their surface expression and channel function. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Photocathode device that replenishes photoemissive coating

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moody, Nathan A.; Lizon, David C.

2016-06-14

A photocathode device may replenish its photoemissive coating to replace coating material that desorbs/evaporates during photoemission. A linear actuator system may regulate the release of a replenishment material vapor, such as an alkali metal, from a chamber inside the photocathode device to a porous cathode substrate. The replenishment material deposits on the inner surface of a porous membrane and effuses through the membrane to the outer surface, where it replenishes the photoemissive coating. The rate of replenishment of the photoemissive coating may be adjusted using the linear actuator system to regulate performance of the photocathode device during photoemission. Alternatively, themore » linear actuator system may adjust a plasma discharge gap between a cartridge containing replenishment material and a metal grid. A potential is applied between the cartridge and the grid, resulting in ejection of metal ions from the cartridge that similarly replenish the photoemissive coating.« less

The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.

PubMed

Wong, Chi-Wai; Lam, Kevin K W; Lee, Cheuk-Lun; Yeung, William S B; Zhao, Wei E; Ho, Pak-Chung; Ou, Jian-Ping; Chiu, Philip C N

2017-04-01

Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction? ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content. A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins. The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed. Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays. Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface expression in vitro stimulated ZP-binding capacity of human spermatozoa. Blocking of ERp57 function by specific antibody or inhibitors against ERp57 reduced the surface thiol content and ZP-binding capacity of human spermatozoa. N/A. The mechanisms by which up-regulation of surface thiol content stimulates spermatozoa-ZP binding have not been depicted. Thiol-disulphide exchange is a crucial event in capacitation. ERp57 modulates the event and the subsequent fertilization process. Modulation of the surface thiol content of the spermatozoa of subfertile men may help to increase fertilization rate in assisted reproduction. This work was supported by The Hong Kong Research Grant Council Grant HKU764611 and HKU764512M to P.C.N.C. The authors have no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Revealing the cell-material interface with nanometer resolution by FIB-SEM

PubMed Central

Santoro, Francesca; Zhao, Wenting; Joubert, Lydia-Marie; Duan, Liting; Schnitker, Jan; van de Burgt, Yoeri; Lou, Hsin-Ya; Liu, Bofei; Salleo, Alberto; Cui, Lifeng; Cui, Yi; Cui, Bianxiao

2018-01-01

The interface between cells and non-biological surfaces regulates cell attachment, chronic tissue responses, and ultimately the success of medical implants or biosensors. Clinical and laboratory studies show that topological features of the surface profoundly influences cellular responses, e.g. titanium surfaces with nano- and microtopographical structures enhance osteoblast attachment and host-implant integration as compare to smooth surface. To understand how cells and tissues respond to different topographical features, it is of critical importance to directly visualize the cell-materials interface at the relevant nanometer length scale. Here, we present a new method for in situ examination of the cell-to-material interface at any desired location, based on focused-ion beam milling and scanning electron microscopy imaging (FIB-SEM) to resolve the cell membrane-to-material interface with 10 nm resolution. By examining how cell membranes interact with topographical features such as nanoscale protrusions or invaginations, we discovered that the cell membrane readily deforms inward and wraps around protruding structures, but hardly deforms outward to contour invaginating structures. This asymmetric membrane response (inward vs. outward deformation) causes the cleft width between the cell membrane and the nanostructure surface to vary for more than an order of magnitude. Our results suggest that surface topology is a crucial consideration for the development of medical implants or biosensors whose performances are strongly influenced by the cell-to-material interface. We anticipate that the method can be used to explore the direct interaction of cells/tissue with medical devices such as metal implants in the future. PMID:28682058

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.

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 importancemore » 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.« less

Isotropic Versus Bipolar Functionalized Biomimetic Artificial Basement Membranes and Their Evaluation in Long-Term Human Cell Co-Culture.

PubMed

Rossi, Angela; Wistlich, Laura; Heffels, Karl-Heinz; Walles, Heike; Groll, Jürgen

2016-08-01

In addition to dividing tissues into compartments, basement membranes are crucial as cell substrates and to regulate cellular behavior. The development of artificial basement membranes is indispensable for the ultimate formation of functional engineered tissues; however, pose a challenge due to their complex structure. Herein, biodegradable electrospun polyester meshes are presented, exhibiting isotropic or bipolar bioactivation as a biomimetic and biofunctional model of the natural basement membrane. In a one-step preparation process, reactive star-shaped prepolymer additives, which generate a hydrophilic fiber surface, are electrospun with cell-adhesion-mediating peptides, derived from major components of the basement membrane. Human skin cells adhere to the functionalized meshes, and long-term co-culture experiments confirm that the artificial basement membranes recapitulate and preserve tissue specific functions. Several layers of immortalized human keratinocytes grow on the membranes, differentiating toward the surface and expressing typical epithelial markers. Fibroblasts migrate into the reticular lamina mimicking part of the mesh. Both cells types begin to produce extracellular matrix proteins and to remodel the initial membrane. It is shown at the example of skin that the artificial basement membrane design provokes biomimetic responses of different cell types and can thus be used as basis for the future development of basement membrane containing tissues. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

Suetsugu, Shiro; Kurisu, Shusaku; Takenawa, Tadaomi

2014-10-01

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

Lubiprostone targets prostanoid signaling and promotes ion transporter trafficking, mucus exocytosis, and contractility.

PubMed

Jakab, Robert L; Collaco, Anne M; Ameen, Nadia A

2012-11-01

Lubiprostone is a chloride channel activator in clinical use for the treatment of chronic constipation, but the mechanisms of action of the drug are poorly understood. The aim of this study was to determine whether lubiprostone exerts secretory effects in the intestine by membrane trafficking of ion transporters and associated machinery. Immunolabeling and quantitative fluorescence intensity were used to examine lubiprostone-induced trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR), sodium/potassium-coupled chloride co-transporter 1 (NKCC1), electrogenic sodium/bicarbonate co-transporter 1 (NBCe1), down-regulated in adenoma (DRA), putative anion transporter 1 (PAT1), sodium/proton exchanger 3 (NHE3), Ca(2+) activated chloride channel 2 (ClC-2) serotonin and its transporter SERT, E prostanoid receptors EP4 and EP1, sodium/potassium ATPase (Na-K-ATPase) and protein kinase A (PKA). The effects of lubiprostone on mucus exocytosis in rat intestine and human rectosigmoid explants were also examined. Lubiprostone induced contraction of villi and proximal colonic plicae and membrane trafficking of transporters that was more pronounced in villus/surface cells compared to the crypt. Membrane trafficking was determined by: (1) increased membrane labeling for CFTR, PAT1, NKCC1, and NBCe1 and decreased membrane labeling for NHE3, DRA and ClC-2; (2) increased serotonin, SERT, EP4, EP1 and PKA labeling in enterochromaffin cells; (3) increased SERT, EP4, EP1, PKA and Na-K-ATPase in enterocytes; and (4) increased mucus exocytosis in goblet cells. These data suggest that lubiprostone can target serotonergic, EP4/PKA and EP1 signaling in surface/villus regions; stimulate membrane trafficking of CFTR/NBCe1/NKCC1 in villus epithelia and PAT1/NBCe1/NKCC1 in colonic surface epithelia; suppress NHE3/DRA trafficking and fluid absorption; and enhance mucus-mobilization and mucosal contractility.

Lubiprostone targets prostanoid signaling and promotes ion transporter trafficking, mucus exocytosis and contractility

PubMed Central

Jakab, Robert L.; Collaco, Anne M.; Ameen, Nadia A.

2012-01-01

Background and Aim Lubiprostone is a chloride channel activator in clinical use for the treatment of chronic constipation, but the mechanisms of action of the drug are poorly understood. The aim of this study was to determine whether lubiprostone exerts secretory effects in the intestine by membrane trafficking of ion transporters and associated machinery. Methods Immunolabeling and quantitative fluorescence intensity were used to examine lubiprostone-induced trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR), sodium/potassium-coupled chloride co-transporter 1 (NKCC1), electrogenic sodium/bicarbonate co-transporter 1 (NBCe1), down-regulated in adenoma (DRA), putative anion transporter 1 (PAT1), sodium/proton exchanger 3 (NHE3), Ca2+ activated chloride channel 2 (ClC-2) serotonin and its transporter SERT, E prostanoid receptors EP4 and EP1, sodium/potassium ATPase (Na-K-ATPase) and protein kinase A (PKA). The effects of lubiprostone on mucus exocytosis in rat intestine and human rectosigmoid explants were also examined. Results Lubiprostone induced contraction of villi and proximal colonic plicae and membrane trafficking of transporters that was more pronounced in villus/surface cells compared to the crypt. Membrane trafficking was determined by: (1) increased membrane labeling for CFTR, PAT1, NKCC1, and NBCe1 and decreased membrane labeling for NHE3, DRA and ClC-2; (2) increased serotonin, SERT, EP4, EP1 and PKA labeling in enterochromaffin cells; (3) increased SERT, EP4, EP1, PKA and Na-K-ATPase in enterocytes; (4) and increased mucus exocytosis in goblet cells. Conclusion These data suggest that lubiprostone can target serotonergic, EP4/PKA and EP1 signaling in surface/villus regions; stimulate membrane trafficking of CFTR/NBCe1/NKCC1 in villus epithelia and PAT1/NBCe1/NKCC1 in colonic surface epithelia; suppress NHE3/DRA trafficking and fluid absorption; enhance mucus-mobilization and mucosal contractility. PMID:22923315

Deconstructing the DGAT1 enzyme: membrane interactions at substrate binding sites.

PubMed

Lopes, Jose L S; Beltramini, Leila M; Wallace, Bonnie A; Araujo, Ana P U

2015-01-01

Diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in the triacylglyceride synthesis pathway. Bovine DGAT1 is an endoplasmic reticulum membrane-bound protein associated with the regulation of fat content in milk and meat. The aim of this study was to evaluate the interaction of DGAT1 peptides corresponding to putative substrate binding sites with different types of model membranes. Whilst these peptides are predicted to be located in an extramembranous loop of the membrane-bound protein, their hydrophobic substrates are membrane-bound molecules. In this study, peptides corresponding to the binding sites of the two substrates involved in the reaction were examined in the presence of model membranes in order to probe potential interactions between them that might influence the subsequent binding of the substrates. Whilst the conformation of one of the peptides changed upon binding several types of micelles regardless of their surface charge, suggesting binding to hydrophobic domains, the other peptide bound strongly to negatively-charged model membranes. This binding was accompanied by a change in conformation, and produced leakage of the liposome-entrapped dye calcein. The different hydrophobic and electrostatic interactions observed suggest the peptides may be involved in the interactions of the enzyme with membrane surfaces, facilitating access of the catalytic histidine to the triacylglycerol substrates.

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells.

PubMed

Kuwada, S K; Lund, K A; Li, X F; Cliften, P; Amsler, K; Opresko, L K; Wiley, H S

1998-12-01

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

P2X7 receptors regulate multiple types of membrane trafficking responses and non-classical secretion pathways.

PubMed

Qu, Yan; Dubyak, George R

2009-06-01

Activation of the P2X7 receptor (P2X7R) triggers a remarkably diverse array of membrane trafficking responses in leukocytes and epithelial cells. These responses result in altered profiles of cell surface lipid and protein composition that can modulate the direct interactions of P2X7R-expressing cells with other cell types in the circulation, in blood vessels, at epithelial barriers, or within sites of immune and inflammatory activation. Additionally, these responses can result in the release of bioactive proteins, lipids, and large membrane complexes into extracellular compartments for remote communication between P2X7R-expressing cells and other cells that amplify or modulate inflammation, immunity, and responses to tissue damages. This review will discuss P2X7R-mediated effects on membrane composition and trafficking in the plasma membrane (PM) and intracellular organelles, as well as actions of P2X7R in controlling various modes of non-classical secretion. It will review P2X7R regulation of: (1) phosphatidylserine distribution in the PM outer leaflet; (2) shedding of PM surface proteins; (3) release of PM-derived microvesicles or microparticles; (4) PM blebbing; (5) cell-cell fusion resulting in formation of multinucleate cells; (6) phagosome maturation and fusion with lysosomes; (7) permeability of endosomes with internalized pathogen-associated molecular patterns; (8) permeability/integrity of mitochondria; (9) exocytosis of secretory lysosomes; and (10) release of exosomes from multivesicular bodies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Chi-Yuan; Kausik, Ravinath; Han, Songi, E-mail: songi@chem.ucsb.edu

Elucidating the physical effect of cholesterol (Chol) on biological membranes is necessary towards rationalizing their structural and functional role in cell membranes. One of the debated questions is the role of hydration water in Chol-embedding lipid membranes, for which only little direct experimental data are available. Here, we study the hydration dynamics in a series of Chol-rich and depleted bilayer systems using an approach termed {sup 1}H Overhauser dynamic nuclear polarization (ODNP) NMR relaxometry that enables the sensitive and selective determination of water diffusion within 5–10 Å of a nitroxide-based spin label, positioned off the surface of the polar headgroupsmore » or within the nonpolar core of lipid membranes. The Chol-rich membrane systems were prepared from mixtures of Chol, dipalmitoyl phosphatidylcholine and/or dioctadecyl phosphatidylcholine lipid that are known to form liquid-ordered, raft-like, domains. Our data reveal that the translational diffusion of local water on the surface and within the hydrocarbon volume of the bilayer is significantly altered, but in opposite directions: accelerated on the membrane surface and dramatically slowed in the bilayer interior with increasing Chol content. Electron paramagnetic resonance (EPR) lineshape analysis shows looser packing of lipid headgroups and concurrently tighter packing in the bilayer core with increasing Chol content, with the effects peaking at lipid compositions reported to form lipid rafts. The complementary capability of ODNP and EPR to site-specifically probe the hydration dynamics and lipid ordering in lipid membrane systems extends the current understanding of how Chol may regulate biological processes. One possible role of Chol is the facilitation of interactions between biological constituents and the lipid membrane through the weakening or disruption of strong hydrogen-bond networks of the surface hydration layers that otherwise exert stronger repulsive forces, as reflected in faster surface water diffusivity. Another is the concurrent tightening of lipid packing that reduces passive, possibly unwanted, diffusion of ions and water across the bilayer.« less

Single Molecule Analysis of Serotonin Transporter Regulation Using Quantum Dots

NASA Astrophysics Data System (ADS)

Chang, Jerry; Tomlinson, Ian; Warnement, Michael; Ustione, Alessandro; Carneiro, Ana; Piston, David; Blakely, Randy; Rosenthal, Sandra

2011-03-01

For the first time, we implement a novel, single molecule approach to define the localization and mobility of the brain's major target of widely prescribed antidepressant medications, the serotonin transporter (SERT). SERT labeled with single quantum dot (Qdot) revealed unsuspected features of transporter mobility with cholesterol-enriched membrane microdomains (often referred to as ``lipid rafts'') and cytoskeleton network linked to transporter activation. We document two pools of surface SERT proteins defined by their lateral mobility, one that exhibits relatively free diffusion in the plasma membrane and a second that displays significantly restricted mobility and localizes to cholesterol-enriched microdomains. Diffusion model prediction and instantaneous velocity analysis indicated that stimuli that act through p38 MAPK-dependent signaling pathways to activate SERT trigger rapid SERT movements within membrane microdomains. Cytoskeleton disruption showed that SERT lateral mobility behaves a membrane raft-constrained, cytoskeleton-associated manner. Our results identify an unsuspected aspect of neurotransmitter transporter regulation that we propose reflects the dissociation of inhibitory, SERT-associated cytoskeletal anchors.

Syndecan-4 Regulates Muscle Differentiation and Is Internalized from the Plasma Membrane during Myogenesis.

PubMed

Rønning, Sissel B; Carlson, Cathrine R; Stang, Espen; Kolset, Svein O; Hollung, Kristin; Pedersen, Mona E

2015-01-01

The cell surface proteoglycan syndecan-4 has been reported to be crucial for muscle differentiation, but the molecular mechanisms still remain to be fully understood. During in vitro differentiation of bovine muscle cells immunocytochemical analyses showed strong labelling of syndecan-4 intracellularly, in close proximity with Golgi structures, in membranes of intracellular vesicles and finally, in the nuclear area including the nuclear envelope. Chase experiments showed that syndecan-4 was internalized from the plasma membrane during this process. Furthermore, when syndecan-4 was knocked down by siRNA more myotubes were formed, and the expression of myogenic transcription factors, β1-integrin and actin was influenced. However, when bovine muscle cells were treated with a cell-penetrating peptide containing the cytoplasmic region of syndecan-4, myoblast fusion and thus myotube formation was blocked, both in normal cells and in syndecan-4 knock down cells. Altogether this suggests that the cytoplasmic domain of syndecan-4 is important in regulation of myogenesis. The internalization of syndecan-4 from the plasma membrane during muscle differentiation and the nuclear localization of syndecan-4 in differentiated muscle cells may be part of this regulation, and is a novel aspect of syndecan biology which merits further studies.

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

PubMed

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

2016-01-01

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

Roles of the multifunctional glycoprotein, emmprin (basigin; CD147), in tumour progression.

PubMed

Yan, Li; Zucker, Stanley; Toole, Bryan P

2005-02-01

Emmprin (basigin;CD147) is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily and is highly enriched on the surface of malignant tumour cells. Emmprin is involved in numerous physiological and pathological systems and exhibits several molecular and cellular characteristics, but a major function of emmprin is stimulation of synthesis of several matrix metalloproteinases. In tumours, emmprin most likely stimulates matrix metalloproteinase production in stromal fibroblasts and endothelial cells as well as in tumour cells themselves by a mechanism involving homophilic interactions between emmprin molecules on apposing cells or on neighbouring cells after membrane vesicle shedding. Membrane-associated cofactors, including caveolin-1 and annexin II, regulate emmprin activity. Emmprin induces angiogenesis via stimulation of VEGF production, invasiveness via stimulation of matrix metalloproteinase production and multidrug resistance via hyaluronan-mediated up-regulation of ErbB2 signaling and cell survival pathway activities. Although the detailed mechanisms whereby it regulates these numerous phenomena are not yet known, it is clear that emmprin is a major mediator of malignant cell behavior.

The Glycosylphosphatidylinositol-PLC in Trypanosoma brucei Forms a Linear Array on the Exterior of the Flagellar Membrane Before and After Activation

PubMed Central

Hanrahan, Orla; Webb, Helena; O'Byrne, Robert; Brabazon, Elaine; Treumann, Achim; Sunter, Jack D.; Carrington, Mark; Voorheis, H. Paul

2009-01-01

Bloodstream forms of Trypanosoma brucei contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) that cleaves the GPI-anchor of the variable surface glycoprotein (VSG). Its location in trypanosomes has been controversial. Here, using confocal microscopy and surface labelling techniques, we show that the GPI-PLC is located exclusively in a linear array on the outside of the flagellar membrane, close to the flagellar attachment zone, but does not co-localize with the flagellar attachment zone protein, FAZ1. Consequently, the GPI-PLC and the VSG occupy the same plasma membrane leaflet, which resolves the topological problem associated with the cleavage reaction if the VSG and the GPI-PLC were on opposite sides of the membrane. The exterior location requires the enzyme to be tightly regulated to prevent VSG release under basal conditions. During stimulated VSG release in intact cells, the GPI-PLC did not change location, suggesting that the release mechanism involves lateral diffusion of the VSG in the plane of the membrane to the fixed position of the GPI-PLC. PMID:19503825

KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells.

PubMed

Garrigues, H Jacques; Rubinchikova, Yelena E; Rose, Timothy M

2014-03-01

Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry. Copyright © 2014 Elsevier Inc. All rights reserved.

The Secretion and Action of Brush Border Enzymes in the Mammalian Small Intestine.

PubMed

Hooton, Diane; Lentle, Roger; Monro, John; Wickham, Martin; Simpson, Robert

2015-01-01

Microvilli are conventionally regarded as an extension of the small intestinal absorptive surface, but they are also, as latterly discovered, a launching pad for brush border digestive enzymes. Recent work has demonstrated that motor elements of the microvillus cytoskeleton operate to displace the apical membrane toward the apex of the microvillus, where it vesiculates and is shed into the periapical space. Catalytically active brush border digestive enzymes remain incorporated within the membranes of these vesicles, which shifts the site of BB digestion from the surface of the enterocyte to the periapical space. This process enables nutrient hydrolysis to occur adjacent to the membrane in a pre-absorptive step. The characterization of BB digestive enzymes is influenced by the way in which these enzymes are anchored to the apical membranes of microvilli, their subsequent shedding in membrane vesicles, and their differing susceptibilities to cleavage from the component membranes. In addition, the presence of active intracellular components of these enzymes complicates their quantitative assay and the elucidation of their dynamics. This review summarizes the ontogeny and regulation of BB digestive enzymes and what is known of their kinetics and their action in the peripheral and axial regions of the small intestinal lumen.

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.

PubMed

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A; Multhoff, Gabriele

2005-06-15

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells

PubMed Central

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A.; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A.

2006-01-01

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo−/CX exosomes were Hsp70 membrane derived positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes. PMID:15958569

Formation of a physiological complex between TRPV2 and RGA protein promotes cell surface expression of TRPV2.

PubMed

Stokes, Alexander J; Wakano, Clay; Del Carmen, Kimberly A; Koblan-Huberson, Murielle; Turner, Helen

2005-03-01

The transient receptor potential, sub-family Vanilloid (TRPV)(2) cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a four-transmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and co-expression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.

Involvement of Rab9 and Rab11 in the intracellular trafficking of TRPC6.

PubMed

Cayouette, Sylvie; Bousquet, Simon M; Francoeur, Nancy; Dupré, Emilie; Monet, Michaël; Gagnon, Hugo; Guedri, Youssef B; Lavoie, Christine; Boulay, Guylain

2010-07-01

TRPC proteins become involved in Ca2+ entry following the activation of Gq-protein coupled receptors. TRPC6 is inserted into the plasma membrane upon stimulation and remains in the plasma membrane as long as the stimulus is present. However, the mechanism that regulates the trafficking of TRPC6 is unclear. In the present study, we highlighted the involvement of two Rab GTPases in the trafficking of TRPC6. Rab9 co-localized in vesicular structures with TRPC6 in HeLa cells and co-immunoprecipitated with TRPC6. When co-expressed with TRPC6, Rab9(S21N), a dominant negative mutant, caused an increase in the level of TRPC6 at the plasma membrane and in TRPC6-mediated Ca2+ entry upon activation by a muscarinic receptor agonist. Similarly, the expression of Rab11 also caused an increase in TRPC6 expression at the cell surface and an increase in TRPC6-mediated Ca2+ entry. The co-expression of TRPC6 with the dominant negative mutant Rab11(S25N) abolished CCh-induced TRPC6 activation and reduced the level of TRPC6 at the plasma membrane. This study demonstrates that the trans-Golgi network and recycling endosomes are involved in the intracellular trafficking of TRPC6 by regulating channel density at the cell surface. 2010 Elsevier B.V. All rights reserved.

Phosphatidic acid modulation of Kv channel voltage sensor function.

PubMed

Hite, Richard K; Butterwick, Joel A; MacKinnon, Roderick

2014-10-06

Membrane phospholipids can function as potent regulators of ion channel function. This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating. Using the method of reconstitution into planar lipid bilayers, in which protein and lipid components are defined and controlled, we characterize two effects of phosphatidic acid. The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces. The second is specific to the presence of a primary phosphate group, acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the voltage sensor. Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the voltage sensor's closed conformation. These findings support a novel mechanism of voltage sensor regulation by the signaling lipid phosphatidic acid.

A central role for vesicle trafficking in epithelial neoplasia: Intracellular highways to carcinogenesis

PubMed Central

Goldenring, James R.

2014-01-01

Epithelial cell carcinogenesis involves the loss of polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation and increased cell motility and invasion. Elements of membrane vesicle trafficking underlie all of these processes. Specific membrane trafficking regulators, including Rab small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine cell surface presentation of proteins and overall function of both differentiated and neoplastic cells. While mutations in vesicle trafficking proteins may not be direct drivers of transformation, elements of the machinery of vesicle movement play critical roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are critical mediators of the full spectrum of cell physiologies driving cancer cell biology, including initial loss of polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may provide important points for manipulation of cancer cell behaviour. PMID:24108097

Radixin regulates synaptic GABAA receptor density and is essential for reversal learning and short-term memory

PubMed Central

Hausrat, Torben J.; Muhia, Mary; Gerrow, Kimberly; Thomas, Philip; Hirdes, Wiebke; Tsukita, Sachiko; Heisler, Frank F.; Herich, Lena; Dubroqua, Sylvain; Breiden, Petra; Feldon, Joram; Schwarz, Jürgen R; Yee, Benjamin K.; Smart, Trevor G.; Triller, Antoine; Kneussel, Matthias

2015-01-01

Neurotransmitter receptor density is a major variable in regulating synaptic strength. Receptors rapidly exchange between synapses and intracellular storage pools through endocytic recycling. In addition, lateral diffusion and confinement exchanges surface membrane receptors between synaptic and extrasynaptic sites. However, the signals that regulate this transition are currently unknown. GABAA receptors containing α5-subunits (GABAAR-α5) concentrate extrasynaptically through radixin (Rdx)-mediated anchorage at the actin cytoskeleton. Here we report a novel mechanism that regulates adjustable plasma membrane receptor pools in the control of synaptic receptor density. RhoA/ROCK signalling regulates an activity-dependent Rdx phosphorylation switch that uncouples GABAAR-α5 from its extrasynaptic anchor, thereby enriching synaptic receptor numbers. Thus, the unphosphorylated form of Rdx alters mIPSCs. Rdx gene knockout impairs reversal learning and short-term memory, and Rdx phosphorylation in wild-type mice exhibits experience-dependent changes when exposed to novel environments. Our data suggest an additional mode of synaptic plasticity, in which extrasynaptic receptor reservoirs supply synaptic GABAARs. PMID:25891999

Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function

PubMed Central

Smith, Gina A.; Fearnley, Gareth W.; Abdul-Zani, Izma; Wheatcroft, Stephen B.; Tomlinson, Darren C.; Harrison, Michael A.

2017-01-01

ABSTRACT Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response. PMID:28798148

Peptide-modified PELCL electrospun membranes for regulation of vascular endothelial cells.

PubMed

Zhou, Fang; Jia, Xiaoling; Yang, Yang; Yang, Qingmao; Gao, Chao; Zhao, Yunhui; Fan, Yubo; Yuan, Xiaoyan

2016-11-01

The efficiency of biomaterials used in small vascular repair depends greatly on their ability to interact with vascular endothelial cells (VECs). Rapid endothelialization of the vascular grafts is a promising way to prevent thrombosis and intimal hyperplasia. In this work, modification of electrospun membranes of poly(ethylene glycol)-b-poly(l-lactide-co-ε-caprolactone) (PELCL) by three different peptides for regulation of VECs were studied in order to obtain ideal bioactive biomaterials as small diameter vascular grafts. QK (a mimetic peptide to vascular endothelial growth factor), Arg-Glu-Asp-Val (REDV, a specific adhesive peptide to VECs) and Val-Ala-Pro-Gly (VAPG, a specific adhesive peptide to vascular smooth muscle cells) were investigated. Surface properties of the modified membranes and the response of VECs were verified. It was found that protein adsorption and platelet adhesion were effectively suppressed with the introduction of QK, REDV or VAPG peptides on the PELCL electrospun membranes. Both QK- and REDV-modified electrospun membranes could accelerate the proliferation of VECs in the first 9days, and the QK-modified electrospun membrane promoted cell proliferation more significantly than the REDV-modified one. The REDV-modified PELCL membrane was the most favorable for VECs adhesion than QK- and VAPG-modified membranes. It was suggested that QK- or REDV-modified PELCL electrospun membranes may have great potential applications in cardiovascular biomaterials for rapid endothelialization in situ. Copyright © 2016 Elsevier B.V. All rights reserved.

Cell Surface Expression of Human Ether-a-go-go-related Gene (hERG) Channels Is Regulated by Caveolin-3 Protein via the Ubiquitin Ligase Nedd4-2*

PubMed Central

Guo, Jun; Wang, Tingzhong; Li, Xian; Shallow, Heidi; Yang, Tonghua; Li, Wentao; Xu, Jianmin; Fridman, Michael D.; Yang, Xiaolong; Zhang, Shetuan

2012-01-01

The human ether-a-go-go-related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (IKr) which plays an important role in cardiac repolarization. A reduction or increase in hERG current can cause long or short QT syndrome, respectively, leading to fatal cardiac arrhythmias. The channel density in the plasma membrane is a key determinant of the whole cell current amplitude. To gain insight into the molecular mechanisms for the regulation of hERG density at the plasma membrane, we used whole cell voltage clamp, Western blotting, and immunocytochemical methods to investigate the effects of an integral membrane protein, caveolin-3 (Cav3) on hERG expression levels. Our data demonstrate that Cav3, hERG, and ubiquitin-ligase Nedd4-2 interact with each other and form a complex. Expression of Cav3 thus enhances the hERG-Nedd4-2 interaction, leading to an increased ubiquitination and degradation of mature, plasma-membrane localized hERG channels. Disrupting Nedd4-2 interaction with hERG by mutations eliminates the effects of Cav3 on hERG channels. Knockdown of endogenous Cav3 or Nedd4-2 in cultured neonatal rat ventricular myocytes using siRNA led to an increase in native IKr. Our data demonstrate that hERG expression in the plasma membrane is regulated by Cav3 via Nedd4-2. These findings extend our understanding of the regulation of hERG channels and cardiac electrophysiology. PMID:22879586

Quantitative Microscopic Analysis of Plasma Membrane Receptor Dynamics in Living Plant Cells.

PubMed

Luo, Yu; Russinova, Eugenia

2017-01-01

Plasma membrane-localized receptors are essential for cellular communication and signal transduction. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 (BRI1) is one of the receptors that is activated by binding to its ligand, the brassinosteroid (BR) hormone, at the cell surface to regulate diverse plant developmental processes. The availability of BRI1 in the plasma membrane is related to its signaling output and is known to be controlled by the dynamic endomembrane trafficking. Advances in fluorescence labeling and confocal microscopy techniques enabled us to gain a better understanding of plasma membrane receptor dynamics in living cells. Here we describe different quantitative microscopy methods to monitor the relative steady-state levels of the BRI1 protein in the plasma membrane of root epidermal cells and its relative exocytosis and recycling rates. The methods can be applied also to analyze similar dynamics of other plasma membrane-localized receptors.

Cholesterol Regulates Multiple Forms of Vesicle Endocytosis at a Mammalian Central Synapse

PubMed Central

Yue, Hai-Yuan; Xu, Jianhua

2015-01-01

Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from nonspecific effects after cholesterol manipulation. Furthermore, it is unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase (COase) or methyl-β-cyclodextrin (MCD) impaired three different forms of endocytosis, i.e., slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca2+ channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of MCD reduced exocytosis, mainly by decreasing the readily releasable pool (RRP) and the vesicle replenishment after RRP depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. PMID:25893258

Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane

PubMed Central

Fujiwara, Takahiro K.; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A.; Watanabe, Yusuke; Umemura, Yasuhiro M.; Murakoshi, Hideji; Suzuki, Kenichi G. N.; Nemoto, Yuri L.; Morone, Nobuhiro; Kusumi, Akihiro

2016-01-01

The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed “hop diffusion”) for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion. PMID:26864625

The neural cell adhesion molecule promotes FGFR-dependent phosphorylation and membrane targeting of the exocyst complex to induce exocytosis in growth cones.

PubMed

Chernyshova, Yana; Leshchyns'ka, Iryna; Hsu, Shu-Chan; Schachner, Melitta; Sytnyk, Vladimir

2011-03-09

The exocyst complex is an essential regulator of polarized exocytosis involved in morphogenesis of neurons. We show that this complex binds to the intracellular domain of the neural cell adhesion molecule (NCAM). NCAM promotes FGF receptor-mediated phosphorylation of two tyrosine residues in the sec8 subunit of the exocyst complex and is required for efficient recruitment of the exocyst complex to growth cones. NCAM at the surface of growth cones induces Ca(2+)-dependent vesicle exocytosis, which is blocked by an inhibitor of L-type voltage-dependent Ca(2+) channels and tetanus toxin. Preferential exocytosis in growth cones underlying neurite outgrowth is inhibited in NCAM-deficient neurons as well as in neurons transfected with phosphorylation-deficient sec8 and dominant-negative peptides derived from the intracellular domain of NCAM. Thus, we reveal a novel role for a cell adhesion molecule in that it regulates addition of the new membrane to the cell surface of growth cones in developing neurons.

Water at Biological Phase Boundaries: Its Role in Interfacial Activation of Enzymes and Metabolic Pathways.

PubMed

Damodaran, Srinivasan

2015-01-01

Many life-sustaining activities in living cells occur at the membrane-water interface. The pertinent questions that we need to ask are, what are the evolutionary reasons in biology for choosing the membrane-water interface as the site for performing and/or controlling crucial biological reactions, and what is the key physical principle that is very singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this chapter, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes through manipulation of the thermodynamic activity of water in the lipid-water interfacial region. The hypothesis is based on the fact that the surface pressure of a lipid monolayer is a direct measure of the thermodynamic activity of water at the lipid-water interface. Accordingly, the surface pressure-dependent activation or inactivation of interfacial enzymes is directly related to changes in the thermodynamic activity of interfacial water. Extension of this argument suggests that cells may manipulate conformations (and activities) of membrane-bound enzymes by manipulating the (re)activity of interfacial water at various locations in the membrane by localized compression or expansion of the interface. In this respect, cells may use the membrane-bound hormone receptors, lipid phase transition, and local variations in membrane lipid composition as effectors of local compression and/or expansion of membrane, and thereby local water activity. Several experimental data in the literature will be reexamined in the light of this hypothesis.

Processing of hemojuvelin requires retrograde trafficking to the Golgi in HepG2 cells.

PubMed

Maxson, Julia E; Enns, Caroline A; Zhang, An-Sheng

2009-02-19

Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with cell membranes through a glycosylphosphatidylinositol anchor or released as a soluble form. Membrane-anchored HJV acts as a coreceptor for bone morphogenetic proteins and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes bone morphogenetic protein signaling and suppresses hepcidin expression. In this study, we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, has complex oligosaccharides and can be derived from HJV with high-mannose oligosaccharides at the plasma membrane. Our results support a model in which retrograde trafficking of HJV before cleavage is the predominant processing pathway. Release of HJV requires it to bind to the transmembrane receptor neogenin. Neogenin does not, however, play a role in HJV trafficking to the cell surface, suggesting that it could be involved either in retrograde trafficking of HJV or in cleavage leading to HJV release.

Processing of hemojuvelin requires retrograde trafficking to the Golgi in HepG2 cells

PubMed Central

Maxson, Julia E.; Enns, Caroline A.

2009-01-01

Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with cell membranes through a glycosylphosphatidylinositol anchor or released as a soluble form. Membrane-anchored HJV acts as a coreceptor for bone morphogenetic proteins and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes bone morphogenetic protein signaling and suppresses hepcidin expression. In this study, we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, has complex oligosaccharides and can be derived from HJV with high-mannose oligosaccharides at the plasma membrane. Our results support a model in which retrograde trafficking of HJV before cleavage is the predominant processing pathway. Release of HJV requires it to bind to the transmembrane receptor neogenin. Neogenin does not, however, play a role in HJV trafficking to the cell surface, suggesting that it could be involved either in retrograde trafficking of HJV or in cleavage leading to HJV release. PMID:19029439

Surface ligand controls silver ion release of nanosilver and its antibacterial activity against Escherichia coli.

PubMed

Long, Yan-Min; Hu, Li-Gang; Yan, Xue-Ting; Zhao, Xing-Chen; Zhou, Qun-Fang; Cai, Yong; Jiang, Gui-Bin

2017-01-01

Understanding the mechanism of nanosilver-dependent antibacterial activity against microorganisms helps optimize the design and usage of the related nanomaterials. In this study, we prepared four kinds of 10 nm-sized silver nanoparticles (AgNPs) with dictated surface chemistry by capping different ligands, including citrate, mercaptopropionic acid, mercaptohexanoic acid, and mercaptopropionic sulfonic acid. Their surface-dependent chemistry and antibacterial activities were investigated. Owing to the weak bond to surface Ag, short carbon chain, and low silver ion attraction, citrate-coated AgNPs caused the highest silver ion release and the strongest antibacterial activity against Escherichia coli , when compared to the other tested AgNPs. The study on the underlying antibacterial mechanisms indicated that cellular membrane uptake of Ag, NAD + /NADH ratio increase, and intracellular reactive oxygen species (ROS) generation were significantly induced in both AgNP and silver ion exposure groups. The released silver ions from AgNPs inside cells through a Trojan-horse-type mechanism were suggested to interact with respiratory chain proteins on the membrane, interrupt intracellular O 2 reduction, and induce ROS production. The further oxidative damages of lipid peroxidation and membrane breakdown caused the lethal effect on E. coli . Altogether, this study demonstrated that AgNPs exerted antibacterial activity through the release of silver ions and the subsequent induction of intracellular ROS generation by interacting with the cell membrane. The findings are helpful in guiding the controllable synthesis through the regulation of surface coating for medical care purpose.

Iron-tannin-framework complex modified PES ultrafiltration membranes with enhanced filtration performance and fouling resistance.

PubMed

Fang, Xiaofeng; Li, Jiansheng; Li, Xin; Pan, Shunlong; Sun, Xiuyun; Shen, Jinyou; Han, Weiqing; Wang, Lianjun; Van der Bruggen, Bart

2017-11-01

In this work, an iron-tannin-framework (ITF) complex was introduced to a poly (ether sulfone) (PES) casting solution as a hydrophilic additive to fabricate ITF/PES ultrafiltration (UF) membranes via non-solvent-induced phase separation (NIPS). The structure and performance of the PES membranes with ITF concentrations ranging from 0 to 0.9wt.% were systematically investigated by scanning electron microscopy, water contact angle, permeability, protein rejection and fouling resistance measurements. The results indicate that the pore structure and surface properties of PES UF membranes can be regulated by incorporating the ITF complex. Compared with classical PES membranes, ITF/PES membranes were found to have an increased hydrophilicity and porosity and reduced surface pore size. Importantly, a simultaneous enhancement of permeability and separation performance was observed for the blend membranes, which indicates that the introduction of the ITF complex can break through the trade-off between permeability and selectivity of UF membranes.When the ITF content was 0.3wt.%, the permeability reached a maximum of 319.4(L/m 2 h) at 0.1MPa, which is 1.6 times higher than that of the classical PES membrane. Furthermore, the BSA rejection increased from 25.9% for the PES membrane to 95.9% for the enhanced membrane. In addition, the same membrane showed an improved fouling resistance (higher flux recovery and lower adhesion force) and stable hydrophilicity (unchanged after incubation in deionized water for 30days). The simple, green and cost-effective preparation process and the outstanding filtration performance highlight the potential of ITF/PES membranes for practical applications. Copyright © 2017 Elsevier Inc. All rights reserved.

Acute ENaC Stimulation by cAMP in a Kidney Cell Line is Mediated by Exocytic Insertion from a Recycling Channel Pool

PubMed Central

Butterworth, Michael B.; Edinger, Robert S.; Johnson, John P.; Frizzell, Raymond A.

2005-01-01

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na+ transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, MPKCCD14) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (ISC), membrane capacitance (CT), and conductance (GT) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 ± 6% (n = 31) increase in ISC and a 15.3 ± 1.5% (n = 15) increase in CT. Selective membrane permeabilization demonstrated that the CT increase was due to an increase in apical membrane capacitance. ISC and CT declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (∼1 h each cycle) resulted in response fatigue; ΔISC decreased ∼10% per stimulation–recovery cycle. When channel production was blocked by cycloheximide, ΔISC decreased ∼15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated ISC, and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the ISC responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation. PMID:15623897

Acute ENaC stimulation by cAMP in a kidney cell line is mediated by exocytic insertion from a recycling channel pool.

PubMed

Butterworth, Michael B; Edinger, Robert S; Johnson, John P; Frizzell, Raymond A

2005-01-01

Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na(+) transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, (MPK)CCD(14)) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (I(SC)), membrane capacitance (C(T)), and conductance (G(T)) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 +/- 6% (n = 31) increase in I(SC) and a 15.3 +/- 1.5% (n = 15) increase in C(T). Selective membrane permeabilization demonstrated that the C(T) increase was due to an increase in apical membrane capacitance. I(SC) and C(T) declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (approximately 1 h each cycle) resulted in response fatigue; DeltaI(SC) decreased approximately 10% per stimulation-recovery cycle. When channel production was blocked by cycloheximide, DeltaI(SC) decreased approximately 15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated I(SC), and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the I(SC) responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.

[Interaction of surface-active base with fraction of membrane-bound Williams's protons].

PubMed

Iaguzhinskiĭ, L S; Motovilov, K A; Volkov, E M; Eremeev, S A

2013-01-01

In the process of mitochondrial respiratory H(+)-pumps functioning, the fraction membrane-bound protons (R-protons), which have an excess of free energy is formed. According to R.J. Williams this fraction is included as energy source in the reaction of ATP synthesis. Previously, in our laboratory was found the formation of this fraction was found in the mitochondria and on the outer surface of mitoplast. On the mitoslast model we strictly shown that non-equilibrium R-proton fraction is localized on the surface of the inner mitochondrial membrane. In this paper a surface-active compound--anion of 2,4,6-trichloro-3-pentadecylphenol (TCP-C15) is described, which selectively interacts with the R-protons fraction in mitochondria. A detailed description of the specific interaction of the TCP-C15 with R-protons fraction in mitochondria is presented. Moreover, in this work it was found that phosphate transport system reacts with the R-protons fraction in mitochondria and plays the role of the endogenous volume regulation system of this fraction. The results of experiments are discussed in the terms of a local coupling model of the phosphorylation mechanism.

High-resolution Structures of Protein-Membrane Complexes by Neutron Reflection and MD Simulation: Membrane Association of the PTEN Tumor Suppressor

NASA Astrophysics Data System (ADS)

Lösche, Matthias

2012-02-01

The lipid matrix of biomembranes is an in-plane fluid, thermally and compositionally disordered leaflet of 5 nm thickness and notoriously difficult to characterize in structural terms. Yet, biomembranes are ubiquitous in the cell, and membrane-bound proteins are implicated in a variety of signaling pathways and intra-cellular transport. We developed methodology to study proteins associated with model membranes using neutron reflection measurements and showed recently that this approach can resolve the penetration depth and orientation of membrane proteins with ångstrom resolution if their crystal or NMR structure is known. Here we apply this technology to determine the membrane bindung and unravel functional details of the PTEN phosphatase, a key player in the PI3K apoptosis pathway. PTEN is an important regulatory protein and tumor suppressor that performs its phosphatase activity as an interfacial enzyme at the plasma membrane-cytoplasm boundary. Acting as an antagonist to phosphoinositide-3-kinase (PI3K) in cell signaling, it is deleted in many human cancers. Despite its importance in regulating the levels of the phosphoinositoltriphosphate PI(3,4,5)P3, there is little understanding of how PTEN binds to membranes, is activated and then acts as a phosphatase. We investigated the structure and function of PTEN by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P2) act synergetically in attracting the enzyme to the membrane surface. Membrane affinities depend strongly on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P2. Neutron reflection measurements show that the PTEN phosphatase ``scoots'' along the membrane surface (penetration < 5 å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains. In the bound state, PTEN's regulatory C-terminal tail is displaced from the membrane and organized on the far side of the protein, ˜ 60 å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN. Molecular dynamics simulations, currently in progress, refine this structural picture further.

Differential Regulation of the Serotonin Transporter by Vesicle-Associated Membrane Protein 2 in Cells of Neuronal versus Non-Neuronal Origin

PubMed Central

Müller, Heidi Kaastrup; Kragballe, Marie; Fjorback, Anja Winther; Wiborg, Ove

2014-01-01

The serotonin transporter (SERT) is a key regulator of serotonergic signalling as it mediates the re-uptake of synaptic serotonin into nerve terminals, thereby terminating or modulating its signal. It is well-known that SERT regulation is a dynamic process orchestrated by a wide array of proteins and mechanisms. However, molecular details on possible coordinated regulation of SERT activity and 5-HT release are incomplete. Here, we report that vesicle-associated membrane protein 2 (VAMP2), a SNARE protein that mediates vesicle fusion with the plasma membrane, interacts with SERT. This was documented in vitro, through GST pull-down assays, by co-immunoprecipitation experiments on heterologous cells and rat hippocampal synaptosomes, and with FRET analysis in live transfected HEK-293 MSR cells. The related isoforms VAMP1 and VAMP3 also physically interact with SERT. However, comparison of the three VAMP isoforms shows that only VAMP2 possesses a functionally distinct role in relation to SERT. VAMP2 influences 5-HT uptake, cell surface expression and the delivery rate of SERT to the plasma membrane differentially in HEK-293 MSR and PC12 cells. Moreover, siRNA-mediated knock-down of endogenous VAMP2 reduces 5-HT uptake in CAD cells stably expressing low levels of heterologous SERT. Deletion and mutant analysis suggest a role for the isoform specific C-terminal domain of VAMP2 in regulating SERT function. Our data identify a novel interaction between SERT and a synaptic vesicle protein and support a link between 5-HT release and re-uptake. PMID:24878716

Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review).

PubMed

Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Brenner, Catherine; Ladant, Daniel; Chopineau, Joël

2017-09-28

Biological membranes and their related molecular mechanisms are essential for all living organisms. Membranes host numerous proteins and are responsible for the exchange of molecules and ions, cell signaling, and cell compartmentation. Indeed, the plasma membrane delimits the intracellular compartment from the extracellular environment and intracellular membranes. Biological membranes also play a major role in metabolism regulation and cellular physiology (e.g., mitochondrial membranes). The elaboration of membrane based biomimetic systems allows us to reconstitute and investigate, in controlled conditions, biological events occurring at the membrane interface. A whole variety of model membrane systems have been developed in the last few decades. Among these models, supported membranes were developed on various hydrophilic supports. The use of solid supports enables the direct use of surface sensitive techniques (e.g., surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy) to monitor and quantify events occurring at the membrane surface. Tethered bilayer membranes (tBLMs) could be considered as an achievement of the first solid supported membranes described by the McConnell group. Tethered bilayers on solid supports were designed to delimit an inside compartment from an outside one. They were used for measuring interactions with ligands or incorporating large membrane proteins or complexes without interference with the support. In this context, the authors developed an easy concept of versatile tBLMs assembled on amino coated substrates that are formed upon the vesicle fusion rupture process applicable to protein-free vesicles as well as proteoliposomes. The phospholipid bilayer (natural or synthetic lipids) incorporated 5% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-N-hydroxy succinimide to ensure the anchorage of the bilayer to the amino coated surface. The conditions for the formation of tBLMs on amino-coated gold and glass were optimized for protein-free vesicles. This biomimetic membrane delimits an inside "trans" compartment separated from an outside reservoir "cis." Using this tBLM construction, the authors were interested in deciphering two complex molecular mechanisms involving membrane-associated proteins. The first one concerns two mitochondrial proteins, i.e., the porin voltage dependent anion channel (VDAC) embedded in the outer membrane and the nucleotide transporter (adenine nucleotide translocase) that interacts dynamically during mitochondrial pathophysiology. The purified VDAC porin was first reconstituted in proteoliposomes that were subsequently assembled on an amino coated support to form a biomimetic membrane. As a major result, VDAC was reconstituted in this tBLM and calcium channeling was demonstrated across the lipid bilayer. The same two-compartment biomimetic membrane design was further engineered to study the translocation mechanism of a bacterial toxin, the adenylate cyclase toxin, CyaA, from Bordetella pertussis. As a result, the authors developed an elegant in vitro translocation toolkit applicable to potentially a large panel of proteins transported across membranes.

Transcriptomic analysis reveals metabolic switches and surface remodeling as key processes for stage transition in Trypanosoma cruzi

PubMed Central

Greif, Gonzalo; Rodriguez, Matias; Alvarez-Valin, Fernando

2017-01-01

American trypanosomiasis is a chronic and endemic disease which affects millions of people. Trypanosoma cruzi, its causative agent, has a life cycle that involves complex morphological and functional transitions, as well as a variety of environmental conditions. This requires a tight regulation of gene expression, which is achieved mainly by post-transcriptional regulation. In this work we conducted an RNAseq analysis of the three major life cycle stages of T. cruzi: amastigotes, epimastigotes and trypomastigotes. This analysis allowed us to delineate specific transcriptomic profiling for each stage, and also to identify those biological processes of major relevance in each state. Stage specific expression profiling evidenced the plasticity of T. cruzi to adapt quickly to different conditions, with particular focus on membrane remodeling and metabolic shifts along the life cycle. Epimastigotes, which replicate in the gut of insect vectors, showed higher expression of genes related to energy metabolism, mainly Krebs cycle, respiratory chain and oxidative phosphorylation related genes, and anabolism related genes associated to nucleotide and steroid biosynthesis; also, a general down-regulation of surface glycoprotein coding genes was seen at this stage. Trypomastigotes, living extracellularly in the bloodstream of mammals, express a plethora of surface proteins and signaling genes involved in invasion and evasion of immune response. Amastigotes mostly express membrane transporters and genes involved in regulation of cell cycle, and also express a specific subset of surface glycoprotein coding genes. In addition, these results allowed us to improve the annotation of the Dm28c genome, identifying new ORFs and set the stage for construction of networks of co-expression, which can give clues about coded proteins of unknown functions. PMID:28286708

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

PubMed Central

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

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS. PMID:27924253

The structure of ions and zwitterionic lipids regulates the charge of dipolar membranes.

PubMed

Szekely, Or; Steiner, Ariel; Szekely, Pablo; Amit, Einav; Asor, Roi; Tamburu, Carmen; Raviv, Uri

2011-06-21

In pure water, zwitterionic lipids form lamellar phases with an equilibrium water gap on the order of 2 to 3 nm as a result of the dominating van der Waals attraction between dipolar bilayers. Monovalent ions can swell those neutral lamellae by a small amount. Divalent ions can adsorb onto dipolar membranes and charge them. Using solution X-ray scattering, we studied how the structure of ions and zwitterionic lipids regulates the charge of dipolar membranes. We found that unlike monovalent ions that weakly interact with all of the examined dipolar membranes, divalent and trivalent ions adsorb onto membranes containing lipids with saturated tails, with an association constant on the order of ∼10 M(-1). One double bond in the lipid tail is sufficient to prevent divalent ion adsorption. We suggest that this behavior is due to the relatively loose packing of lipids with unsaturated tails that increases the area per lipid headgroup, enabling their free rotation. Divalent ion adsorption links two lipids and limits their free rotation. The ion-dipole interaction gained by the adsorption of the ions onto unsaturated membranes is insufficient to compensate for the loss of headgroup free-rotational entropy. The ion-dipole interaction is stronger for cations with a higher valence. Nevertheless, polyamines behave as monovalent ions near dipolar interfaces in the sense that they interact weakly with the membrane surface, whereas in the bulk their behavior is similar to that of multivalent cations. Advanced data analysis and comparison with theory provide insight into the structure and interactions between ion-induced regulated charged interfaces. This study models biologically relevant interactions between cell membranes and various ions and the manner in which the lipid structure governs those interactions. The ability to monitor these interactions creates a tool for probing systems that are more complex and forms the basis for controlling the interactions between dipolar membranes and charged proteins or biopolymers for encapsulation and delivery applications. © 2011 American Chemical Society

Internal and Surface-Localized Major Surface Proteases of Leishmania spp. and Their Differential Release from Promastigotes▿

PubMed Central

Yao, Chaoqun; Donelson, John E.; Wilson, Mary E.

2007-01-01

Major surface protease (MSP), also called GP63, is a virulence factor of Leishmania spp. protozoa. There are three pools of MSP, located either internally within the parasite, anchored to the surface membrane, or released into the extracellular environment. The regulation and biological functions of these MSP pools are unknown. We investigated here the trafficking and extrusion of surface versus internal MSPs. Virulent Leishmania chagasi undergo a growth-associated lengthening in the t1/2 of surface-localized MSP, but this did not occur in the attenuated L5 strain. The release of surface-localized MSP was enhanced in a dose-dependent manner by MβCD, which chelates membrane cholesterol-ergosterol. Furthermore, incubation of promastigotes at 37°C with Matrigel matrix, a soluble basement membrane extract of Engelbreth-Holm-Swarm tumor cells, stimulated the release of internal MSP but not of surface-located MSP. Taken together, these data indicate that MSP subpopulations in distinct cellular locations are released from the parasite under different environmental conditions. We hypothesize that the internal MSP with its lengthy t1/2 does not serve as a pool for promastigote surface MSP in the sand fly vector but that it instead functions as an MSP pool ready for quick release upon inoculation of metacyclic promastigotes into mammals. We present a model in which these different MSP pools are released under distinct life cycle-specific conditions. PMID:17693594

Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1

PubMed Central

Teriete, Peter; Thai, Khang; Choi, Jungyuen; Marassi, Francesca M.

2009-01-01

FXYD1 (phospholemman) is a member of an evolutionarily conserved family of membrane proteins that regulate the function of the Na,K-ATPase enzyme complex in specific tissues and specific physiological states. In heart and skeletal muscle sarcolemma, FXYD1 is also the principal substrate of hormone-regulated phosphorylation by c-AMP dependent protein kinase A and by protein kinase C, which phosphorylate the protein at conserved Ser residues in its cytoplasmic domain, altering its Na,K-ATPase regulatory activity. FXYD1 adopts an L-shaped α-helical structure with the transmembrane helix loosely connected to a cytoplasmic amphipathic helix that rests on the membrane surface. In this paper we describe NMR experiments showing that neither PKA phosphorylation at Ser68 nor the physiologically relevant phosphorylation mimicking mutation Ser68Asp induces major changes in the protein conformation. The results, viewed in light of a model of FXYD1 associated with the Na,K-ATPase α and β subunits, indicate that the effects of phosphorylation on the Na,K-ATPase regulatory activity of FXYD1 could be due primarily to changes in electrostatic potential near the membrane surface and near the Na+/K+ ion binding site of the Na,K-ATPase α subunit. PMID:19761758

Structural Basis of Membrane Targeting by the Dock180 Family of Rho Family Guanine Exchange Factors (Rho-GEFs)*

PubMed Central

Premkumar, Lakshmanane; Bobkov, Andrey A.; Patel, Manishha; Jaroszewski, Lukasz; Bankston, Laurie A.; Stec, Boguslaw; Vuori, Kristiina; Côté, Jean-Francois; Liddington, Robert C.

2010-01-01

The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P3 head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of the Rho-GEF activity of Dock180. PMID:20167601

The G-protein coupled estrogen receptor, GPER: The inside and inside-out story.

PubMed

Gaudet, H M; Cheng, S B; Christensen, E M; Filardo, E J

2015-12-15

GPER possesses structural and functional characteristics shared by members of the G-protein-coupled receptor (GPCR) superfamily, the largest class of plasma membrane receptors. This newly appreciated estrogen receptor is localized predominately within intracellular membranes in most, but not all, cell types and its surface expression is modulated by steroid hormones and during tissue injury. An intracellular staining pattern is not unique among GPCRs, which employ a diverse array of molecular mechanisms that restrict cell surface expression and effectively regulating receptor binding and activation. The finding that GPER displays an intracellular predisposition has created some confusion as the estrogen-inducible transcription factors, ERα and ERβ, also reside intracellularly, and has led to complex suggestions of receptor interaction. GPER undergoes constitutive retrograde trafficking from the plasma membrane to the endoplasmic reticulum and recent studies indicate its interaction with PDZ binding proteins that sort transmembrane receptors to synaptosomes and endosomes. Genetic targeting and selective ligand approaches as well as cell models that express GPER in the absence of ERs clearly supports GPER as a bonafide "stand alone" receptor. Here, the molecular details that regulate GPER action, its cell biological activities and its implicated roles in physiological and pathological processes are reviewed. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

PubMed Central

Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

2014-01-01

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

Plasma membrane--cortical cytoskeleton interactions: a cell biology approach with biophysical considerations.

PubMed

Kapus, András; Janmey, Paul

2013-07-01

From a biophysical standpoint, the interface between the cell membrane and the cytoskeleton is an intriguing site where a "two-dimensional fluid" interacts with an exceedingly complex three-dimensional protein meshwork. The membrane is a key regulator of the cytoskeleton, which not only provides docking sites for cytoskeletal elements through transmembrane proteins, lipid binding-based, and electrostatic interactions, but also serves as the source of the signaling events and molecules that control cytoskeletal organization and remolding. Conversely, the cytoskeleton is a key determinant of the biophysical and biochemical properties of the membrane, including its shape, tension, movement, composition, as well as the mobility, partitioning, and recycling of its constituents. From a cell biological standpoint, the membrane-cytoskeleton interplay underlies--as a central executor and/or regulator--a multitude of complex processes including chemical and mechanical signal transduction, motility/migration, endo-/exo-/phagocytosis, and other forms of membrane traffic, cell-cell, and cell-matrix adhesion. The aim of this article is to provide an overview of the tight structural and functional coupling between the membrane and the cytoskeleton. As biophysical approaches, both theoretical and experimental, proved to be instrumental for our understanding of the membrane/cytoskeleton interplay, this review will "oscillate" between the cell biological phenomena and the corresponding biophysical principles and considerations. After describing the types of connections between the membrane and the cytoskeleton, we will focus on a few key physical parameters and processes (force generation, curvature, tension, and surface charge) and will discuss how these contribute to a variety of fundamental cell biological functions. © 2013 American Physiological Society.

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

PubMed Central

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

2016-01-01

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

Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

PubMed

Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

2015-11-19

Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor internalization-induced changes in neuronal functions of the CNS.

Multiple roles for the actin cytoskeleton during regulated exocytosis

PubMed Central

Porat-Shliom, Natalie; Milberg, Oleg; Masedunskas, Andrius; Weigert, Roberto

2014-01-01

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules. PMID:22986507

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

PubMed

Nishimura, Tamako; Morone, Nobuhiro; Suetsugu, Shiro

2018-04-17

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

Cytosolic proteins can exploit membrane localization to trigger functional assembly

PubMed Central

2018-01-01

Cell division, endocytosis, and viral budding would not function without the localization and assembly of protein complexes on membranes. What is poorly appreciated, however, is that by localizing to membranes, proteins search in a reduced space that effectively drives up concentration. Here we derive an accurate and practical analytical theory to quantify the significance of this dimensionality reduction in regulating protein assembly on membranes. We define a simple metric, an effective equilibrium constant, that allows for quantitative comparison of protein-protein interactions with and without membrane present. To test the importance of membrane localization for driving protein assembly, we collected the protein-protein and protein-lipid affinities, protein and lipid concentrations, and volume-to-surface-area ratios for 46 interactions between 37 membrane-targeting proteins in human and yeast cells. We find that many of the protein-protein interactions between pairs of proteins involved in clathrin-mediated endocytosis in human and yeast cells can experience enormous increases in effective protein-protein affinity (10–1000 fold) due to membrane localization. Localization of binding partners thus triggers robust protein complexation, suggesting that it can play an important role in controlling the timing of endocytic protein coat formation. Our analysis shows that several other proteins involved in membrane remodeling at various organelles have similar potential to exploit localization. The theory highlights the master role of phosphoinositide lipid concentration, the volume-to-surface-area ratio, and the ratio of 3D to 2D equilibrium constants in triggering (or preventing) constitutive assembly on membranes. Our simple model provides a novel quantitative framework for interpreting or designing in vitro experiments of protein complexation influenced by membrane binding. PMID:29505559

Regulation of STIM1 and SOCE by the ubiquitin-proteasome system (UPS).

PubMed

Keil, Jeffrey M; Shen, Zhouxin; Briggs, Steven P; Patrick, Gentry N

2010-10-18

The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca(2+) entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3's), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca(2+) homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function.

Phosphatidic acid modulation of Kv channel voltage sensor function

PubMed Central

Hite, Richard K; Butterwick, Joel A; MacKinnon, Roderick

2014-01-01

Membrane phospholipids can function as potent regulators of ion channel function. This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating. Using the method of reconstitution into planar lipid bilayers, in which protein and lipid components are defined and controlled, we characterize two effects of phosphatidic acid. The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces. The second is specific to the presence of a primary phosphate group, acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the voltage sensor. Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the voltage sensor's closed conformation. These findings support a novel mechanism of voltage sensor regulation by the signaling lipid phosphatidic acid. DOI: http://dx.doi.org/10.7554/eLife.04366.001 PMID:25285449

Cadherin 99C regulates apical expansion and cell rearrangement during epithelial tube elongation

PubMed Central

Chung, SeYeon; Andrew, Deborah J.

2014-01-01

Apical and basolateral determinants specify and maintain membrane domains in epithelia. Here, we identify new roles for two apical surface proteins – Cadherin 99C (Cad99C) and Stranded at Second (SAS) – in conferring apical character in Drosophila tubular epithelia. Cad99C, the Drosophila ortholog of human Usher protocadherin PCDH15, is expressed in several embryonic tubular epithelial structures. Through loss-of-function and overexpression studies, we show that Cad99C is required to regulate cell rearrangement during salivary tube elongation. We further show that overexpression of either Cad99C or SAS causes a dramatic increase in apical membrane at the expense of other membrane domains, and that both proteins can do this independently of each other and independently of mislocalization of the apical determinant Crumbs (Crb). Overexpression of Cad99C or SAS results in similar, but distinct effects, suggesting both shared and unique roles for these proteins in conferring apical identity. PMID:24718992

The Rcs-Regulated Colanic Acid Capsule Maintains Membrane Potential in Salmonella enterica serovar Typhimurium

PubMed Central

Pando, Jasmine M.; Karlinsey, Joyce E.; Lara, Jimmie C.; Libby, Stephen J.

2017-01-01

ABSTRACT The Rcs phosphorelay and Psp (phage shock protein) systems are envelope stress responses that are highly conserved in gammaproteobacteria. The Rcs regulon was found to be strongly induced during metal deprivation of Salmonella enterica serovar Typhimurium lacking the Psp response. Nineteen genes activated by the RcsA-RcsB response regulator make up an operon responsible for the production of colanic acid capsular polysaccharide, which promotes biofilm development. Despite more than half a century of research, the physiological function of colanic acid has remained elusive. Here we show that Rcs-dependent colanic acid production maintains the transmembrane electrical potential and proton motive force in cooperation with the Psp response. Production of negatively charged exopolysaccharide covalently bound to the outer membrane may enhance the surface potential by increasing the local proton concentration. This provides a unifying mechanism to account for diverse Rcs/colanic acid-related phenotypes, including susceptibility to membrane-damaging agents and biofilm formation. PMID:28588134

Cholesterol regulates multiple forms of vesicle endocytosis at a mammalian central synapse.

PubMed

Yue, Hai-Yuan; Xu, Jianhua

2015-07-01

Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from non-specific effects after cholesterol manipulation. Furthermore, it remains unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase or methyl-β-cyclodextrin impaired three different forms of endocytosis, including slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca(2+) channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of methyl-β-cyclodextrin reduced exocytosis, mainly by decreasing the readily releasable pool and the vesicle replenishment after readily releasable pool depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses. © 2015 International Society for Neurochemistry.

Review: Post-translational cross-talk between brassinosteroid and sucrose signaling.

PubMed

Kühn, Christina

2016-07-01

A direct link has been elucidated between brassinosteroid function and perception, and sucrose partitioning and transport. Sucrose regulation and brassinosteroid signaling cross-talk at various levels, including the well-described regulation of transcriptional gene expression: BZR-like transcription factors link the signaling pathways. Since brassinosteroid responses depend on light quality and quantity, a light-dependent alternative pathway was postulated. Here, the focus is on post-translational events. Recent identification of sucrose transporter-interacting partners raises the question whether brassinosteroid and sugars jointly affect plant innate immunity and plant symbiotic interactions. Membrane permeability and sensitivity depends on the number of cell surface receptors and transporters. More than one endocytic route has been assigned to specific components, including brassinosteroid-receptors. The number of such proteins at the plasma membrane relies on endocytic recycling, internalization and/or degradation. Therefore, vesicular membrane trafficking is gaining considerable attention with regard to plant immunity. The organization of pattern recognition receptors (PRRs), other receptors or transporters in membrane microdomains participate in endocytosis and the formation of specific intracellular compartments, potentially impacting biotic interactions. This minireview focuses on post-translational events affecting the subcellular compartmentation of membrane proteins involved in signaling, transport, and defense, and on the cross-talk between brassinosteroid signals and sugar availability. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Zwitterionic materials for antifouling membrane surface construction.

PubMed

He, Mingrui; Gao, Kang; Zhou, Linjie; Jiao, Zhiwei; Wu, Mengyuan; Cao, Jialin; You, Xinda; Cai, Ziyi; Su, Yanlei; Jiang, Zhongyi

2016-08-01

Membrane separation processes are often perplexed by severe and ubiquitous membrane fouling. Zwitterionic materials, keeping electric neutrality with equivalent positive and negative charged groups, are well known for their superior antifouling properties and have been broadly utilized to construct antifouling surfaces for medical devices, biosensors and marine coatings applications. In recent years, zwitterionic materials have been more and more frequently utilized for constructing antifouling membrane surfaces. In this review, the antifouling mechanisms of zwitterionic materials as well as their biomimetic prototypes in cell membranes will be discussed, followed by the survey of common approaches to incorporate zwitterionic materials onto membrane surfaces including surface grafting, surface segregation, biomimetic adhesion, surface coating and so on. The potential applications of these antifouling membranes are also embedded. Finally, we will present a brief perspective on the future development of zwitterionic materials modified antifouling membranes. Membrane fouling is a severe problem hampering the application of membrane separation technology. The properties of membrane surfaces play a critical role in membrane fouling and antifouling behavior/performance. Antifouling membrane surface construction has evolved as a hot research issue for the development of membrane processes. Zwitterionic modification of membrane surfaces has been recognized as an effective strategy to resist membrane fouling. This review summarizes the antifouling mechanisms of zwitterionic materials inspired by cell membranes as well as the popular approaches to incorporate them onto membrane surfaces. It can help form a comprehensive knowledge about the principles and methods of modifying membrane surfaces with zwitterionic materials. Finally, we propose the possible future research directions of zwitterionic materials modified antifouling membranes. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Regulation of the Epithelial-Mesenchymal Transition in Prostate Cancer

DTIC Science & Technology

2013-06-01

removed by a cotton swab, and the cells on the lower surface of the membrane were stained by crystal violet. BD BioCoat Matrigel Invasion Chambers...PTK6 downstream players, including AKT, p130CAS, FAK, and ERK5. AKT signaling pro- motes the EMT in different cancer cell lines (23, 45, 46). PTK6...Yang R, Crawford SE, Vasioukhin V, Fuchs E, et al. Protein tyrosine kinase 6 negatively regulates growth and pro- motes enterocyte differentiation in

CD147, CD44, and the epidermal growth factor receptor (EGFR) signaling pathway cooperate to regulate breast epithelial cell invasiveness.

PubMed

Grass, G Daniel; Tolliver, Lauren B; Bratoeva, Momka; Toole, Bryan P

2013-09-06

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777-788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.

Layer-by-Layer Assembly for Preparation of High-Performance Forward Osmosis Membrane

NASA Astrophysics Data System (ADS)

Yang, Libin; Zhang, Jinglong; Song, Peng; Wang, Zhan

2018-01-01

Forward osmosis (FO) membrane with high separation performance is needed to promote its practical applications. Herein, layer-by-layer (LbL) approach was used to prepare a thin and highly cross-linked polyamide layer on a polyacrylonitrile substrate surface to prepare a thin-film composite forward osmosis (TFC-FO) membrane with enhanced FO performance. The effects of monomer concentrations and assembly cycles on the performance of the TFC-FO membranes were systematically investigated. Under the optimal preparation condition, TFC-FO membrane achieved the best performance, exhibiting the water flux of 14.4/6.9 LMH and reverse salt flux of 7.7/3.8 gMH under the pressure retarded osmosis/forward osmosis (PRO/FO) mode using 1M NaCl as the draw against a DI-water feed, and a rejection of 96.1% for 2000 mg/L NaCl aqueous solution. The result indicated that layer-by-layer method was a potential method to regulate the structure and performance of the TFC-FO membrane.

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle

PubMed Central

Peterson, Soren J.; Krasnow, Mark A.

2015-01-01

SUMMARY To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains including inside the cell. PMID:25557078

Subcellular trafficking of FGF controls tracheal invasion of Drosophila flight muscle.

PubMed

Peterson, Soren J; Krasnow, Mark A

2015-01-15

To meet the extreme oxygen demand of insect flight muscle, tracheal (respiratory) tubes ramify not only on its surface, as in other tissues, but also within T-tubules and ultimately surrounding every mitochondrion. Although this remarkable physiological specialization has long been recognized, its cellular and molecular basis is unknown. Here, we show that Drosophila tracheoles invade flight muscle T-tubules through transient surface openings. Like other tracheal branching events, invasion requires the Branchless FGF pathway. However, localization of the FGF chemoattractant changes from all muscle membranes to T-tubules as invasion begins. Core regulators of epithelial basolateral membrane identity localize to T-tubules, and knockdown of AP-1γ, required for basolateral trafficking, redirects FGF from T-tubules to surface, increasing tracheal surface ramification and preventing invasion. We propose that tracheal invasion is controlled by an AP-1-dependent switch in FGF trafficking. Thus, subcellular targeting of a chemoattractant can direct outgrowth to specific domains, including inside the cell. Copyright © 2015 Elsevier Inc. All rights reserved.

Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

PubMed Central

2012-01-01

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96. PMID:22292497

Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

PubMed

Weekes, Michael P; Antrobus, Robin; Talbot, Suzanne; Hör, Simon; Simecek, Nikol; Smith, Duncan L; Bloor, Stuart; Randow, Felix; Lehner, Paul J

2012-03-02

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

Engineering Surface Energy and Nanostructure of Microporous Films for Expanded Membrane Distillation Applications.

PubMed

Boo, Chanhee; Lee, Jongho; Elimelech, Menachem

2016-08-02

We investigated the factors that determine surface omniphobicity of microporous membranes and evaluated the potential application of these membranes in desalination of low surface tension wastewaters by membrane distillation (MD). Specifically, the effects of surface morphology and surface energy on membrane surface omniphobicity were systematically investigated by evaluating wetting resistance to low surface tension liquids. Single and multilevel re-entrant structures were achieved by using cylindrical glass fibers as a membrane substrate and grafting silica nanoparticles (SiNPs) on the fibers. Surface energy of the membrane was tuned by functionalizing the fiber substrate with fluoroalkylsilane (FAS) having two different lengths of fluoroalkyl chains. Results show that surface omniphobicity of the modified fibrous membrane increased with higher level of re-entrant structure and with lower surface energy. The secondary re-entrant structure achieved by SiNP coating on the cylindrical fibers was found to play a critical role in enhancing the surface omniphobicity. Membranes coated with SiNPs and chemically modified by the FAS with a longer fluoroalkyl chain (or lower surface energy) exhibited excellent surface omniphobicity and showed wetting resistance to low surface tension liquids such as ethanol (22.1 mN m(-1)). We further evaluated performance of the membranes in desalination of saline feed solutions with varying surface tensions by membrane distillation (MD). The engineered membranes exhibited stable MD performance with low surface tension feed waters, demonstrating the potential application omniphobic membranes in desalinating complex, high salinity industrial wastewaters.

The Rheological Properties of Lipid Monolayers Modulate the Incorporation of l-Ascorbic Acid Alkyl Esters.

PubMed

Díaz, Yenisleidy de Las Mercedes Zulueta; Mottola, Milagro; Vico, Raquel V; Wilke, Natalia; Fanani, María Laura

2016-01-19

In this work, we tested the hypothesis that the incorporation of amphiphilic drugs into lipid membranes may be regulated by their rheological properties. For this purpose, two members of the l-ascorbic acid alkyl esters family (ASCn) were selected, ASC16 and ASC14, which have different rheological properties when organized at the air/water interface. They are lipophilic forms of vitamin C used in topical pharmacological preparations. The effect of the phase state of the host lipid membranes on ASCn incorporation was explored using Langmuir monolayers. Films of pure lipids with known phase states have been selected, showing liquid-expanded, liquid-condensed, and solid phases as well as pure cholesterol films in liquid-ordered state. We also tested ternary and quaternary mixed films that mimic the properties of cholesterol containing membranes and of the stratum corneum. The compressibility and shear properties of those monolayers were assessed in order to define its phase character. We found that the length of the acyl chain of the ASCn compounds induces differential changes in the rheological properties of the host membrane and subtly regulates the kinetics and extent of the penetration process. The capacity for ASCn uptake was found to depend on the phase state of the host film. The increase in surface pressure resultant after amphiphile incorporation appears to be a function of the capacity of the host membrane to incorporate such amphiphile as well as the rheological response of the film. Hence, monolayers that show a solid phase state responded with a larger surface pressure increase to the incorporation of a comparable amount of amphiphile than liquid-expanded ones. The cholesterol-containing films, including the mixture that mimics stratum corneum, allowed a very scarce ASCn uptake independently of the membrane diffusional properties. This suggests an important contribution of Cho on the maintenance of the barrier function of stratum corneum.

Photo-triggered release from liposomes without membrane solubilization, based on binding to poly(vinyl alcohol) carrying a malachite green moiety.

PubMed

Uda, Ryoko M; Kato, Yutaka; Takei, Michiko

2016-10-01

When working with liposomes analogous to cell membranes, it is important to develop substrates that can regulate interactions with the liposome surface in response to light. We achieved a photo-triggered release from liposomes by using a copolymer of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG). Although PVAMG is a neutral polymer under dark conditions, it is photoionized upon exposure to UV light, resulting in the formation of a cationic site for binding to liposomes with a negatively charged surface. Under UV irradiation, PVAMG showed effective interaction with liposomes, releasing the encapsulated compound; however, this release was negligible under dark conditions. The poly(vinyl alcohol) moiety of PVAMG played an important role in the photo-triggered release. This release was caused by membrane destabilization without lipid solubilization. We also investigated different aspects of liposome/PVAMG interactions, including PVAMG-induced fusion between the liposomes and the change in the liposome morphologies. Copyright © 2016 Elsevier B.V. All rights reserved.

MinD-dependent conformational changes in MinE required for the Min oscillator to spatially regulate cytokinesis

PubMed Central

Park, Kyung-Tae; Wu, Wei; Battaile, Kevin P.; Lovell, Scott; Holyoak, Todd; Lutkenhaus, Joe

2011-01-01

Summary MinD recruits MinE to the membrane leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring in E. coli. How these proteins interact, however, is not clear since the MinD binding regions of MinE are sequestered within a 6-stranded β-sheet and masked by N-terminal helices. Here, minE mutations are isolated that restore interaction to some MinD and MinE mutants. These mutations alter the MinE structure releasing the MinD binding regions and N-terminal helices that bind MinD and the membrane, respectively. Crystallization of MinD-MinE complexes reveals a 4-stranded β-sheet MinE dimer with the released β strands (MinD binding regions) converted to α-helices bound to MinD dimers. These results suggest a 6 stranded, β-sheet dimer of MinE ‘senses’ MinD and switches to a 4-stranded β-sheet dimer that binds MinD and contributes to membrane binding. Also, the results indicate how MinE persists at the MinD-membrane surface. PMID:21816275

Biophysical Model of Ion Transport across Human Respiratory Epithelia Allows Quantification of Ion Permeabilities

PubMed Central

Garcia, Guilherme J.M.; Boucher, Richard C.; Elston, Timothy C.

2013-01-01

Lung health and normal mucus clearance depend on adequate hydration of airway surfaces. Because transepithelial osmotic gradients drive water flows, sufficient hydration of the airway surface liquid depends on a balance between ion secretion and absorption by respiratory epithelia. In vitro experiments using cultures of primary human nasal epithelia and human bronchial epithelia have established many of the biophysical processes involved in airway surface liquid homeostasis. Most experimental studies, however, have focused on the apical membrane, despite the fact that ion transport across respiratory epithelia involves both cellular and paracellular pathways. In fact, the ion permeabilities of the basolateral membrane and paracellular pathway remain largely unknown. Here we use a biophysical model for water and ion transport to quantify ion permeabilities of all pathways (apical, basolateral, paracellular) in human nasal epithelia cultures using experimental (Ussing Chamber and microelectrode) data reported in the literature. We derive analytical formulas for the steady-state short-circuit current and membrane potential, which are for polarized epithelia the equivalent of the Goldman-Hodgkin-Katz equation for single isolated cells. These relations allow parameter estimation to be performed efficiently. By providing a method to quantify all the ion permeabilities of respiratory epithelia, the model may aid us in understanding the physiology that regulates normal airway surface hydration. PMID:23442922

Metabotropic glutamate receptor 5 upregulates surface NMDA receptor expression in striatal neurons via CaMKII.

PubMed

Jin, Dao-Zhong; Xue, Bing; Mao, Li-Min; Wang, John Q

2015-10-22

Metabotropic and ionotropic glutamate receptors are closely clustered in postsynaptic membranes and are believed to interact actively with each other to control excitatory synaptic transmission. Metabotropic glutamate receptor 5 (mGluR5), for example, has been well documented to potentiate ionotropic NMDA receptor activity, although underlying mechanisms are poorly understood. In this study, we investigated the role of mGluR5 in regulating trafficking and subcellular distribution of NMDA receptors in adult rat striatal neurons. We found that the mGluR1/5 agonist DHPG concentration-dependently increased NMDA receptor GluN1 and GluN2B subunit expression in the surface membrane. Meanwhile, DHPG reduced GluN1 and GluN2B levels in the intracellular compartment. The effect of DHPG was blocked by an mGluR5 selective antagonist MTEP but not by an mGluR1 selective antagonist 3-MATIDA. Pretreatment with an inhibitor or a specific inhibitory peptide for synapse-enriched Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) also blocked the DHPG-stimulated redistribution of GluN1 and GluN2B. In addition, DHPG enhanced CaMKIIα activity and elevated GluN2B phosphorylation at a CaMKII-sensitive site (serine 1303). These results demonstrate that mGluR5 regulates trafficking of NMDA receptors in striatal neurons. Activation of mGluR5 appears to induce rapid trafficking of GluN1 and GluN2B to surface membranes through a signaling pathway involving CaMKII. Copyright © 2015 Elsevier B.V. All rights reserved.

Presynaptic Type III Neuregulin1-ErbB signaling targets α7 nicotinic acetylcholine receptors to axons

PubMed Central

Hancock, Melissa L.; Canetta, Sarah E.; Role, Lorna W.; Talmage, David A.

2008-01-01

Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of α7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface α7 nAChRs, which results from a redistribution of preexisting intracellular pools of α7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting α7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function. PMID:18458158

Presynaptic type III neuregulin1-ErbB signaling targets {alpha}7 nicotinic acetylcholine receptors to axons.

PubMed

Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

2008-05-05

Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

Presynaptic type III neuregulin1-ErbB signaling targets alpha7 nicotinic acetylcholine receptors to axons.

PubMed

Hancock, Melissa L; Canetta, Sarah E; Role, Lorna W; Talmage, David A

2008-06-01

Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of alpha7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface alpha7 nAChRs, which results from a redistribution of preexisting intracellular pools of alpha7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting alpha7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney.

PubMed

Guggino, W B; Oberleithner, H; Giebisch, G

1985-07-01

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway.

Relationship between cell volume and ion transport in the early distal tubule of the Amphiuma kidney

PubMed Central

1985-01-01

The roles of apical and basolateral transport mechanisms in the regulation of cell volume and the hydraulic water permeabilities (Lp) of the individual cell membranes of the Amphiuma early distal tubule (diluting segment) were evaluated using video and optical techniques as well as conventional and Cl-sensitive microelectrodes. The Lp of the apical cell membrane calculated per square centimeter of tubule is less than 3% that of the basolateral cell membrane. Calculated per square centimeter of membrane, the Lp of the apical cell membrane is less than 40% that of the basolateral cell membrane. Thus, two factors are responsible for the asymmetry in the Lp of the early distal tubule: an intrinsic difference in the Lp per square centimeter of membrane area, and a difference in the surface areas of the apical and basolateral cell membranes. Early distal tubule cells do not regulate volume after a reduction in bath osmolality. This cell swelling occurs without a change in the intracellular Cl content or the basolateral cell membrane potential. In contrast, reducing the osmolality of the basolateral solution in the presence of luminal furosemide diminishes the magnitude of the increase in cell volume to a value below that predicted from the change in osmolality. This osmotic swelling is associated with a reduction in the intracellular Cl content. Hence, early distal tubule cells can lose solute in response to osmotic swelling, but only after the apical Na/K/Cl transporter is blocked. Inhibition of basolateral Na/K ATPase with ouabain results in severe cell swelling. This swelling in response to ouabain can be inhibited by the prior application of furosemide, which suggests that the swelling is due to the continued entry of solutes, primarily through the apical cotransport pathway. PMID:2411847

Arf and Rho GAP adapter protein ARAP1 participates in the mobilization of TRAIL-R1/DR4 to the plasma membrane.

PubMed

Símová, Sárka; Klíma, Martin; Cermak, Lukas; Sourková, Vladimíra; Andera, Ladislav

2008-03-01

TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.

Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

PubMed Central

Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

2011-01-01

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

Multiple facets of sialomucin complex/MUC4, a membrane mucin and erbb2 ligand, in tumors and tissues (Y2K update).

PubMed

Carraway, K L; Price-Schiavi, S A; Komatsu, M; Idris, N; Perez, A; Li, P; Jepson, S; Zhu, X; Carvajal, M E; Carraway, C A

2000-01-01

Sialomucin complex (SMC, MUC4) is a high Mr glycoprotein heterodimer, composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. ASGP-2 contains two EGF-like domains and acts as an intramembrane ligand for the receptor tyrosine kinase ErbB2. Transfection studies with SMC DNAs showed that SMC expression could markedly reduce both cell-cell and cell-matrix interactions in vitro and increase the growth of primary tumors and the formation of metastatic foci of human A375 melanoma cells as xenotransplants in nude mice, possibly through the ability to suppress apoptosis. SMC is expressed in most vulnerable epithelia as a protective agent, which is found in both membrane and soluble forms at luminal surfaces and secreted into fluids such as milk and tears. SMC appears to be constitutively expressed by most accessible epithelia, notable exceptions being the mammary gland and uterine luminal epithelium, in which it is tightly regulated during pregnancy. Down-regulation at the luminal uterine surface appears necessary for blastocyst implantation. TGF-b is a potent repressor of SMC expression in the mammary gland and uterus, though by different mechanisms. These combined results suggest that SMC has multiple functions in epithelia and is tightly regulated in those tissues where its special functions are required.

Robust hydrophobic polyurethane fibrous membranes with tunable porous structure for waterproof and breathable application

NASA Astrophysics Data System (ADS)

Gu, Jiatai; Gu, Haihong; Cao, Jin; Chen, Shaojie; Li, Ni; Xiong, Jie

2018-05-01

In this work, novel nanofibrous membranes with waterproof and breathable (W&B) performance were successfully fabricated by the combination of electrospinning and surface modification technology. This fibrous membranes consisted of polyurethane (PU), NaCl, and fluoroalkylsilane (FAS). Firstly, The fibrous construction and porous structure of fibrous membranes were regulated by tuning the NaCl concentrations in PU solutions. Then, the obtained PU/NaCl fibrous membranes were further modified with fluoroalkylsilane (FAS) to improve hydrophobic property. The synergistic effect of porous structure and hydrophobicity on waterproof and breathable performance was investigated. Furthermore, the mechanical property of fibrous membranes was deeply analysed on the basis of macromolecule orientation and adhesive structure. Benefiting from the optimized porous structure and hydrophobic modification, the resultant fibrous membranes exhibited excellent waterproof (hydrostatic pressure of 1261 Mbar), breathable (water vapor transmission (WVT) rate of 9.06 kg m-2 d-1 and air permeability of 4.8 mm s-1) performance, as well as high tensile strength (breakage stress of 10.4 MPa), suggesting a promising candidate for various applications, especially in protective clothing.

Plasma Membrane Factor XIIIA Transglutaminase Activity Regulates Osteoblast Matrix Secretion and Deposition by Affecting Microtubule Dynamics

PubMed Central

Al-Jallad, Hadil F.; Myneni, Vamsee D.; Piercy-Kotb, Sarah A.; Chabot, Nicolas; Mulani, Amina; Keillor, Jeffrey W.; Kaartinen, Mari T.

2011-01-01

Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to ‘block –and-track’ enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics. PMID:21283799

Mapping cell surface adhesion by rotation tracking and adhesion footprinting

NASA Astrophysics Data System (ADS)

Li, Isaac T. S.; Ha, Taekjip; Chemla, Yann R.

2017-03-01

Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.

Superficial physicochemical properties of polyurethane biomaterials as osteogenic regulators in human mesenchymal stem cells fates.

PubMed

Shahrousvand, Mohsen; Sadeghi, Gity Mir Mohamad; Shahrousvand, Ehsan; Ghollasi, Marzieh; Salimi, Ali

2017-08-01

All of the cells' interactions are done through their surfaces. Evaluation of surface physicochemical scaffolds along with other factors is important and determines the fate of stem cells. In this work, biodegradable and biocompatible polyester/polyether based polyurethanes (PUs) were synthesized by polycaprolactone diol (PCL) and poly (tetra methylene ether) glycol (PTMEG) as the soft segment. To assess better the impact of surface parameters such as stiffness and roughness effects on osteogenic differentiation of the human mesenchymal stem cell (hMSC), the dimension effect of substrates was eliminated and two-dimensional membranes were produced by synthesized polyurethane. Surface and bulk properties of prepared 2D membranes such as surface chemistry, roughness, stiffness and tensile behavior were evaluated by Attenuated total reflectance Fourier transform infrared (ATR-FTIR), atomic force microscopy (AFM) and tensile behavior. The prepared 2D PU films had suitable hydrophilicity, biodegradability, water absorption, surface roughness and bulk strength. The hMSCs showed greater osteogenesis expression in PU substrates with more roughness and stiffness than others. The results demonstrated that surface parameters along with other differentiation cues have a synergistic effect on stem cells fates. Copyright © 2017 Elsevier B.V. All rights reserved.

Determinants of GPI-PLC Localisation to the Flagellum and Access to GPI-Anchored Substrates in Trypanosomes

PubMed Central

Sunter, Jack; Webb, Helena; Carrington, Mark

2013-01-01

In Trypanosoma brucei, glycosylphosphatidylinositol phospholipase C (GPI-PLC) is a virulence factor that releases variant surface glycoprotein (VSG) from dying cells. In live cells, GPI-PLC is localised to the plasma membrane where it is concentrated on the flagellar membrane, so activity or access must be tightly regulated as very little VSG is shed. Little is known about regulation except that acylation within a short internal motif containing three cysteines is necessary for GPI-PLC to access VSG in dying cells. Here, GPI-PLC mutants have been analysed both for subcellular localisation and for the ability to release VSG from dying cells. Two sequence determinants necessary for concentration on the flagellar membrane were identified. First, all three cysteines are required for full concentration on the flagellar membrane. Mutants with two cysteines localise predominantly to the plasma membrane but lose some of their flagellar concentration, while mutants with one cysteine are mainly localised to membranes between the nucleus and flagellar pocket. Second, a proline residue close to the C-terminus, and distant from the acylated cysteines, is necessary for concentration on the flagellar membrane. The localisation of GPI-PLC to the plasma but not flagellar membrane is necessary for access to the VSG in dying cells. Cellular structures necessary for concentration on the flagellar membrane were identified by depletion of components. Disruption of the flagellar pocket collar caused loss of concentration whereas detachment of the flagellum from the cell body after disruption of the flagellar attachment zone did not. Thus, targeting to the flagellar membrane requires: a titratable level of acylation, a motif including a proline, and a functional flagellar pocket. These results provide an insight into how the segregation of flagellar membrane proteins from those present in the flagellar pocket and cell body membranes is achieved. PMID:23990786

Mechanisms regulating cell membrane localization of the chemokine receptor CXCR4 in human hepatocarcinoma cells.

PubMed

Cepeda, Edgar B; Dediulia, Tatjana; Fernando, Joan; Bertran, Esther; Egea, Gustavo; Navarro, Estanislao; Fabregat, Isabel

2015-05-01

Hepatocellular carcinoma (HCC) cells with a mesenchymal phenotype show an asymmetric subcellular distribution of the chemokine receptor CXCR4, which is required for cell migration and invasion. In this work we examine the mechanisms that regulate the intracellular trafficking of CXCR4 in HCC cells. Results indicate that HCC cells present CXCR4 at the cell surface, but most of this protein is in endomembranes colocalizing with markers of the Golgi apparatus and recycling endosomes. The presence of high protein levels of CXCR4 present at the cell surface correlates with a mesenchymal-like phenotype and a high autocrine activation of the Transforming Growth Factor-beta (TGF-β) pathway. CXCR4 traffics along the Golgi/exocyst/plasma membrane pathway and requires EXOC4 (Sec8) component of the exocyst complex. HCC cells use distinct mechanisms for the CXCR4 internalization such as dynamin-dependent endocytosis and macropinocytosis. Regardless of the endocytic mechanisms, colocalization of CXCR4 and Rab11 is observed, which could be involved not only in receptor recycling but also in its post-Golgi transport. In summary, this work highlights membrane trafficking pathways whose pharmacological targeting could subsequently result in the inactivation of one of the main guiding mechanisms used by metastatic cells to colonize secondary organs and tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

Dynamic complexity: plant receptor complexes at the plasma membrane.

PubMed

Burkart, Rebecca C; Stahl, Yvonne

2017-12-01

Plant receptor complexes at the cell surface perceive many different external and internal signalling molecules and relay these signals into the cell to regulate development, growth and immunity. Recent progress in the analyses of receptor complexes using different live cell imaging approaches have shown that receptor complex formation and composition are dynamic and take place at specific microdomains at the plasma membrane. In this review we focus on three prominent examples of Arabidopsis thaliana receptor complexes and how their dynamic spatio-temporal distribution at the PM has been studied recently. We will elaborate on the newly emerging concept of plasma membrane microdomains as potential hubs for specific receptor complex assembly and signalling outputs. Copyright © 2017 Elsevier Ltd. All rights reserved.

STEAP: A prostate-specific cell-surface antigen highly expressed in human prostate tumors

PubMed Central

Hubert, Rene S.; Vivanco, Igor; Chen, Emily; Rastegar, Shiva; Leong, Kahan; Mitchell, Steve C.; Madraswala, Rashida; Zhou, Yanhong; Kuo, James; Raitano, Arthur B.; Jakobovits, Aya; Saffran, Douglas C.; Afar, Daniel E. H.

1999-01-01

In search of novel genes expressed in metastatic prostate cancer, we subtracted cDNA isolated from benign prostatic hypertrophic tissue from cDNA isolated from a prostate cancer xenograft model that mimics advanced disease. One novel gene that is highly expressed in advanced prostate cancer encodes a 339-amino acid protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl-terminal domains. This structure suggests a potential function as a channel or transporter protein. This gene, named STEAP for six-transmembrane epithelial antigen of the prostate, is expressed predominantly in human prostate tissue and is up-regulated in multiple cancer cell lines, including prostate, bladder, colon, ovarian, and Ewing sarcoma. Immunohistochemical analysis of clinical specimens demonstrates significant STEAP expression at the cell–cell junctions of the secretory epithelium of prostate and prostate cancer cells. Little to no staining was detected at the plasma membranes of normal, nonprostate human tissues, except for bladder tissue, which expressed low levels of STEAP at the cell membrane. Protein analysis located STEAP at the cell surface of prostate-cancer cell lines. Our results support STEAP as a cell-surface tumor-antigen target for prostate cancer therapy and diagnostic imaging. PMID:10588738

Iodide handling by the thyroid epithelial cell.

PubMed

Nilsson, M

2001-01-01

Iodination of thyroglobulin, the key event in the synthesis of thyroid hormone, is an extracellular process that takes place inside the thyroid follicles at the apical membrane surface that faces the follicular lumen. The supply of iodide involves two steps of TSH-regulated transport, basolateral uptake and apical efflux, that imprint the polarized phenotype of the thyroid cell. Iodide uptake is generated by the sodium/iodide symporter present in the basolateral plasma membrane. A candidate for the apical iodide-permeating mechanism is pendrin, a chloride/iodide transporting protein recently identified in the apical membrane. In physiological conditions, transepithelial iodide transport occurs without intracellular iodination, despite the presence of large amounts of thyroglobulin and thyroperoxidase inside the cells. The reason is that hydrogen peroxide, serving as electron acceptor in iodide-protein binding and normally produced at the apical cell surface, is rapidly degraded by cytosolic glutathione peroxidase once it enters the cells. Iodinated thyroglobulin in the lumen stores not only thyroid hormone but iodine incorporated in iodotyrosine residues as well. After endocytic uptake and degradation of thyroglobulin, intracellular deiodination provides a mechanism for recycling of iodide to participate in the synthesis of new thyroid hormone at the apical cell surface.

Polymeric membranes: surface modification for minimizing (bio)colloidal fouling.

PubMed

Kochkodan, Victor; Johnson, Daniel J; Hilal, Nidal

2014-04-01

This paper presents an overview on recent developments in surface modification of polymer membranes for reduction of their fouling with biocolloids and organic colloids in pressure driven membrane processes. First, colloidal interactions such as London-van der Waals, electrical, hydration, hydrophobic, steric forces and membrane surface properties such as hydrophilicity, charge and surface roughness, which affect membrane fouling, have been discussed and the main goals of the membrane surface modification for fouling reduction have been outlined. Thereafter the recent studies on reduction of (bio)colloidal of polymer membranes using ultraviolet/redox initiated surface grafting, physical coating/adsorption of a protective layer on the membrane surface, chemical reactions or surface modification of polymer membranes with nanoparticles as well as using of advanced atomic force microscopy to characterize (bio)colloidal fouling have been critically summarized. Copyright © 2013 Elsevier B.V. All rights reserved.

Characterization of Surface Modification of Polyethersulfone Membrane

USDA-ARS?s Scientific Manuscript database

Surface modification of polyethersulfone (PES) membrane surface using UV/ozone-treated grafting and interfacial polymerization on membrane surface was investigated in order to improve the resistance of membrane surface to protein adsorption. These methods of surface modification were compared in te...

Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity.

PubMed

Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

2015-09-01

Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity[S

PubMed Central

Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

2015-01-01

Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. PMID:26175473

Proteomic analysis of enterotoxigenic Escherichia coli (ETEC) in neutral and alkaline conditions.

PubMed

Gonzales-Siles, Lucia; Karlsson, Roger; Kenny, Diarmuid; Karlsson, Anders; Sjöling, Åsa

2017-01-07

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea in children and travelers to endemic areas. Secretion of the heat labile AB 5 toxin (LT) is induced by alkaline conditions. In this study, we determined the surface proteome of ETEC exposed to alkaline conditions (pH 9) as compared to neutral conditions (pH 7) using a LPI Hexalane FlowCell combined with quantitative proteomics. Relative quantitation with isobaric labeling (TMT) was used to compare peptide abundance and their corresponding proteins in multiple samples at MS/MS level. For protein identification and quantification samples were analyzed using either a 1D-LCMS or a 2D-LCMS approach. Strong up-regulation of the ATP synthase operon encoding F1Fo ATP synthase and down-regulation of proton pumping proteins NuoF, NuoG, Ndh and WrbA were detected among proteins involved in regulating the proton and electron transport under alkaline conditions. Reduced expression of proteins involved in osmotic stress was found at alkaline conditions while the Sec-dependent transport over the inner membrane and outer membrane protein proteins such as OmpA and the β-Barrel Assembly Machinery (BAM) complex were up-regulated. ETEC exposed to alkaline environments express a specific proteome profile characterized by up-regulation of membrane proteins and secretion of LT toxin. Alkaline microenvironments have been reported close to the intestinal epithelium and the alkaline proteome may hence represent a better view of ETEC during infection.

Using a biomimetic membrane surface experiment to investigate the activity of the magnetite biomineralisation protein Mms6† †Electronic supplementary information (ESI) available: Including Mms6 protein and peptide sequences, additional QCM-D and SEM data and protein modelling. See DOI: 10.1039/c5ra16469a Click here for additional data file.

PubMed Central

Bird, Scott M.; Rawlings, Andrea E.; Galloway, Johanna M.

2016-01-01

Magnetotactic bacteria are able to synthesise precise nanoparticles of the iron oxide magnetite within their cells. These particles are formed in dedicated organelles termed magnetosomes. These lipid membrane compartments use a range of biomineralisation proteins to nucleate and regulate the magnetite crystallisation process. A key component is the membrane protein Mms6, which binds to iron ions and helps to control the formation of the inorganic core. We have previously used Mms6 on gold surfaces patterned with a self-assembled monolayer to successfully produce arrays of magnetic nanoparticles. Here we use this surface system as a mimic of the interior face of the magnetosome membrane to study differences between intact Mms6 and the acid-rich C-terminal peptide subregion of the Mms6 protein. When immobilised on surfaces, the peptide is unable to reproduce the particle size or homogeneity control exhibited by the full Mms6 protein in our experimental setup. Moreover, the peptide is unable to support anchoring of a dense array of nanoparticles to the surface. This system also allows us to deconvolute particle binding from particle nucleation, and shows that Mms6 particle binding is less efficient when supplied with preformed magnetite nanoparticles when compared to particles precipitated from solution in the presence of the surface immobilised Mms6. This suggests that Mms6 binds to iron ions rather than to magnetite surfaces in our system, and is perhaps a nucleating agent rather than a controller of magnetite crystal growth. The comparison between the peptide and the protein under identical experimental conditions indicates that the full length sequence is required to support the full function of Mms6 on surfaces. PMID:27019707

Effects of fiber density and plasma modification of nanofibrous membranes on the adhesion and growth of HaCaT keratinocytes.

PubMed

Bacakova, Marketa; Lopot, Frantisek; Hadraba, Daniel; Varga, Marian; Zaloudkova, Margit; Stranska, Denisa; Suchy, Tomas; Bacakova, Lucie

2015-01-01

It may be possible to regulate the cell colonization of biodegradable polymer nanofibrous membranes by plasma treatment and by the density of the fibers. To test this hypothesis, nanofibrous membranes of different fiber densities were treated by oxygen plasma with a range of plasma power and exposure times. Scanning electron microscopy and mechanical tests showed significant modification of nanofibers after plasma treatment. The intensity of the fiber modification increased with plasma power and exposure time. The exposure time seemed to have a stronger effect on modifying the fiber. The mechanical behavior of the membranes was influenced by the plasma treatment, the fiber density, and their dry or wet state. Plasma treatment increased the membrane stiffness; however, the membranes became more brittle. Wet membranes displayed significantly lower stiffness than dry membranes. X-ray photoelectron spectroscopy (XPS) analysis showed a slight increase in oxygen-containing groups on the membrane surface after plasma treatment. Plasma treatment enhanced the adhesion and growth of HaCaT keratinocytes on nanofibrous membranes. The cells adhered and grew preferentially on membranes of lower fiber densities, probably due to the larger area of void spaces between the fibers. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells.

PubMed

Ehre, Camille; Rossi, Andrea H; Abdullah, Lubna H; De Pestel, Kathleen; Hill, Sandra; Olsen, John C; Davis, C William

2005-01-01

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G(q)-coupled P2Y(2) receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of beta- and gamma-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of beta- or gamma-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca(2+)-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

An approach to the research on ion and water properties in the interphase between the plasma membrane and bulk extracellular solution.

PubMed

Hibino, Hiroshi; Takai, Madoka; Noguchi, Hidenori; Sawamura, Seishiro; Takahashi, Yasufumi; Sakai, Hideki; Shiku, Hitoshi

2017-07-01

In vivo, cells are immersed in an extracellular solution that contains a variety of bioactive substances including ions and water. Classical electrophysiological analyses of epithelial cells in the stomach and small intestine have revealed that within a distance of several hundred micrometers above their apical plasma membrane, lies an extracellular layer that shows ion concentration gradients undetectable in the bulk phase. This "unstirred layer", which contains stagnant solutes, may also exist between the bulk extracellular solution and membranes of other cells in an organism and may show different properties. On the other hand, an earlier study using a bacterial planar membrane indicated that H + released from a transporter migrates in the horizontal direction along the membrane surface much faster than it diffuses vertically toward the extracellular space. This result implies that between the membrane surface and unstirred layer, there is a "nanointerface" that has unique ionic dynamics. Advanced technologies have revealed that the nanointerface on artificial membranes possibly harbors a highly ordered assembly of water molecules. In general, hydrogen bonds are involved in formation of the ordered water structure and can mediate rapid transfer of H + between neighboring molecules. This description may match the phenomenon on the bacterial membrane. A recent study has suggested that water molecules in the nanointerface regulate the gating of K + channels. Here, the region comprising the unstirred layer and nanointerface is defined as the interphase between the plasma membrane and bulk extracellular solution (iMES). This article briefly describes the physicochemical properties of ions and water in the iMES and their physiological significance. We also describe the methodologies that are currently used or will be applicable to the interphase research.

Endosomal sorting of GLUT4 and Gap1 is conserved between yeast and insulin-sensitive cells

PubMed Central

Shewan, Annette M.; McCann, Rebecca K.; Lamb, Christopher A.; Stirrat, Laura; Kioumourtzoglou, Dimitrios; Adamson, Iain S.; Verma, Suzie; James, David E.; Bryant, Nia J.

2013-01-01

Summary The insulin-regulated trafficking of the facilitative glucose transporter GLUT4 in human fat and muscle cells and the nitrogen-regulated trafficking of the general amino acid permease Gap1 in the yeast Saccharomyces cerevisiae share several common features: Both Gap1 and GLUT4 are nutrient transporters that are mobilised to the cell surface from an intracellular store in response to an environmental cue; both are polytopic membrane proteins harbouring amino acid targeting motifs in their C-terminal tails that are required for their regulated trafficking; ubiquitylation of both Gap1 and GLUT4 plays an important role in their regulated trafficking, as do the ubiquitin-binding GGA (Golgi-localised, γ-ear-containing, ARF-binding) adaptor proteins. Here, we find that when expressed heterologously in yeast, human GLUT4 is subject to nitrogen-regulated trafficking in an ubiquitin-dependent manner similar to Gap1. In addition, by expressing a GLUT4/Gap1 chimeric protein in adipocytes we show that the carboxy-tail of Gap1 directs intracellular sequestration and insulin-regulated trafficking in adipocytes. These findings demonstrate that the trafficking signals and their cognate molecular regulatory machinery that mediate regulated exocytosis of membrane proteins are conserved across evolution. PMID:23424197

Relating Silica Scaling in Reverse Osmosis to Membrane Surface Properties.

PubMed

Tong, Tiezheng; Zhao, Song; Boo, Chanhee; Hashmi, Sara M; Elimelech, Menachem

2017-04-18

We investigated the relationship between membrane surface properties and silica scaling in reverse osmosis (RO). The effects of membrane hydrophilicity, free energy for heterogeneous nucleation, and surface charge on silica scaling were examined by comparing thin-film composite polyamide membranes grafted with a variety of polymers. Results show that the rate of silica scaling was independent of both membrane hydrophilicity and free energy for heterogeneous nucleation. In contrast, membrane surface charge demonstrated a strong correlation with the extent of silica scaling (R 2 > 0.95, p < 0.001). Positively charged membranes significantly facilitated silica scaling, whereas a more negative membrane surface charge led to reduced scaling. This observation suggests that deposition of negatively charged silica species on the membrane surface plays a critical role in silica scale formation. Our findings provide fundamental insights into the mechanisms governing silica scaling in reverse osmosis and highlight the potential of membrane surface modification as a strategy to reduce silica scaling.

Single Molecule Analysis of Serotonin Transporter Regulation Using Antagonist-Conjugated Quantum Dots Reveals Restricted, p38 MAPK-Dependent Mobilization Underlying Uptake Activation

PubMed Central

Chang, Jerry C.; Tomlinson, Ian D.; Warnement, Michael R.; Ustione, Alessandro; Carneiro, Ana M. D.; Piston, David W.; Blakely, Randy D.; Rosenthal, Sandra J.

2012-01-01

The presynaptic serotonin (5-HT) transporter (SERT) is targeted by widely prescribed antidepressant medications. Altered SERT expression or regulation has been implicated in multiple neuropsychiatric disorders, including anxiety, depression and autism. Here, we implement a generalizable strategy that exploits antagonist-conjugated quantum dots (Qdots) to monitor, for the first time, single SERT proteins on the surface of serotonergic cells. We document two pools of SERT proteins defined by lateral mobility, one that exhibits relatively free diffusion, and a second, localized to cholesterol and GM1 ganglioside-enriched microdomains, that displays restricted mobility. Receptor-linked signalling pathways that enhance SERT activity mobilize transporters that, nonetheless, remain confined to membrane microdomains. Mobilization of transporters arise from a p38 MAPK-dependent untethering of the SERT C-terminus from the juxtamembrane actin cytoskeleton. Our studies establish the utility of ligand-conjugated Qdots for analysis of the behaviour of single membrane proteins and reveal a physical basis for signaling-mediated SERT regulation. PMID:22745492

The hypertonic environment differentially regulates wild-type CFTR and TNR-CFTR chloride channels.

PubMed

Lassance-Soares, Roberta M; Cheng, Jie; Krasnov, Kristina; Cebotaru, Liudmila; Cutting, Garry R; Souza-Menezes, Jackson; Morales, Marcelo M; Guggino, William B

2010-01-01

This study tested the hypotheses that the hypertonic environment of the renal medulla regulates the expression of cystic fibrosis transmembrane conductance regulator protein (CFTR) and its natural splice variant, TNR-CFTR. To accomplish this, Madin-Darby canine kidney (MDCK) stable cell lines expressing TNR-CFTR or CFTR were used. The cells were treated with hypertonic medium made with either NaCl or urea or sucrose (480 mOsm/kg or 560 mOsm/kg) to mimic the tonicity of the renal medulla environment. Western blot data showed that CFTR and TNR-CFTR total cell protein is increased by hypertonic medium, but using the surface biotinylation technique, only CFTR was found to be increased in cell plasma membrane. Confocal microscopy showed TNR-CFTR localization primarily at the endoplasmic reticulum and plasma membrane. In conclusion, CFTR and TNR-CFTR have different patterns of distribution in MDCK cells and they are modulated by a hypertonic environment, suggesting their physiological importance in renal medulla. Copyright © 2010 S. Karger AG, Basel.

Oxygen-­dependent regulation of bacterial lipid production

DOE PAGES

Lemmer, Kimberly C.; Dohnalkova, Alice C.; Noguera, Daniel R.; ...

2015-05-02

Understanding the mechanisms of lipid accumulation in microorganisms is important for several reasons. In addition to providing insight into assembly of biological membranes, lipid accumulation has important applications in the production of renewable fuels and chemicals. The photosynthetic bacterium Rhodobacter sphaeroides is an attractive organism to study lipid accumulation, as it has the somewhat unique ability to increase membrane production at low O₂ tensions. Under these conditions, R. sphaeroides develops invaginations of the cytoplasmic membrane to increase its membrane surface area for housing of the membrane-bound components of its photosynthetic apparatus. Here we use fatty acid levels as a reportermore » of membrane lipid content. We show that, under low-O₂ and anaerobic conditions, the total fatty acid content per cell increases 3-fold. We also find that the increases in the amount of fatty acid and photosynthetic pigment per cell are correlated as O₂ tensions or light intensity are changed. To ask if lipid and pigment accumulation were genetically separable, we analyzed strains with mutations in known photosynthetic regulatory pathways. While a strain lacking AppA failed to induce photosynthetic pigment-protein complex accumulation, it increased fatty acid content under low O2 conditions. We also found that an intact PrrBA pathway is required for low O2-induced fatty acid accumulation. In conclusion, our findings suggest a previously unknown role of R. sphaeroides transcriptional regulators in increasing fatty acid and phospholipid accumulation in response to decreased O₂ tension.« less

Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking.

PubMed

Mayor, S; Rothberg, K G; Maxfield, F R

1994-06-24

Glycosyl-phosphatidylinositol (GPI)-anchored proteins have been reported to reside in clusters collected over small membrane invaginations called caveolae. The detection of different GPI-anchored proteins with fluorescently labeled monoclonal antibodies showed that these proteins are not constitutively concentrated in caveolae; they enter these structures independently after cross-linking with polyclonal secondary antibodies. Analysis of the cell surface distribution of the GPI-anchored folate receptor by electron microscopy confirms these observations. Thus, multimerization of GPI-anchored proteins regulates their sequestration in caveolae, but in the absence of agents that promote clustering they are diffusely distributed over the plasma membrane.

CD70 is downregulated by interaction with CD27

PubMed Central

Kuka, Mirela; Munitic, Ivana; Torchia, Maria Letizia Giardino; Ashwell, Jonathan D.

2013-01-01

Engagement of the receptor CD27 by CD70 affects the magnitude and quality of T cell responses in a variety of infection models, and exaggerated signaling via this pathway results in enhanced immune responses and autoimmunity. One means by which signaling is regulated is tight control of cell surface CD70, which is expressed on dendritic, T, and B cells only upon activation. Here we show that there is a second level of regulation. First, although undetectable on the cell surface by flow cytometry, immature dendritic cells (DC) have a small pool of CD70 that continuously recycles from the plasma membrane. In addition, surface levels of CD70 on DC and T cells were higher in mice deficient in CD27, or on DC for which the interaction between CD70 and CD27 was precluded by blocking antibodies. Binding of CD70 by its receptor resulted in downregulation of CD70 transcription and protein levels, suggesting that CD70-mediated “reverse signals” regulate its own levels. Therefore, the ability of CD70 to trigger costimulation is self-regulated when it binds its complementary receptor. PMID:23913967

Pore channel surface modification for enhancing anti-fouling membrane distillation

NASA Astrophysics Data System (ADS)

Qiu, Haoran; Peng, Yuelian; Ge, Lei; Villacorta Hernandez, Byron; Zhu, Zhonghua

2018-06-01

Membrane surface modification by forming a functional layer is an effective way to improve the anti-fouling properties of membranes; however, the additional layer and the potential blockage of bulk pores may increase the mass transfer resistance and reduce the permeability. In this study, we applied a novel method of preparing anti-fouling membranes for membrane distillation by dispersing graphene oxide (GO) on the channel surface of polyvinylidene fluoride membranes. The surface morphology and properties were characterized by scanning electron microscopy, atomic force microscope, and Fourier transform infrared spectrometry. Compared to the membrane surface modification by nanoparticles (e.g. SiO2), GO was mainly located on the pore surface of the membrane bulk, rather than being formed as an individual layer onto the membrane surface. The performance was evaluated via a direct-contact membrane distillation process with anionic and cationic surfactants as the foulants, separately. Compared to the pristine PVDF membrane, the anti-fouling behavior and distillate flux of the GO-modified membranes were improved, especially when using the anionic surfactant as the foulant. The enhanced anti-fouling performance can be attributed to the oxygen containing functional groups in GO and the healing of the membrane pore defects. This method may provide an effective route to manipulate membrane pore surface properties for anti-fouling separation without increasing mass transfer resistance.

Membrane-Based Water Evaporator for a Space Suit

NASA Technical Reports Server (NTRS)

Ungar, Eugene K.; McCann, Charles J.; O'Connell, Mary K.; Andrea, Scott

2004-01-01

A membrane-based water evaporator has been developed that is intended to serve as a heat-rejection device for a space suit. This evaporator would replace the current sublimator that is sensitive to contamination of its feedwater. The design of the membrane-based evaporator takes advantage of recent advances in hydrophobic micropore membranes to provide robust heat rejection with much less sensitivity to contamination. The low contamination sensitivity allows use of the heat transport loop as feedwater, eliminating the need for the separate feedwater system used for the sublimator. A cross section of the evaporator is shown in the accompanying figure. The space-suit cooling loop water flows into a distribution plenum, through a narrow annulus lined on both sides with a hydrophobic membrane, into an exit plenum, and returns to the space suit. Two perforated metal tubes encase the membranes and provide structural strength. Evaporation at the membrane inner surface dissipates the waste heat from the space suit. The water vapor passes through the membrane, into a steam duct and is vented to the vacuum environment through a back-pressure valve. The back-pressure setting can be adjusted to regulate the heat-rejection rate and the water outlet temperature.

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

PubMed Central

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

2016-01-01

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

Trafficking and Membrane Organization of GPI-Anchored Proteins in Health and Diseases.

PubMed

Paladino, Simona; Lebreton, Stéphanie; Zurzolo, Chiara

2015-01-01

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are a class of lipid-anchored proteins attached to the membranes by a glycolipid anchor that is added, as posttranslation modification, in the endoplasmic reticulum. GPI-APs are expressed at the cell surface of eukaryotes where they play diverse vital functions. Like all plasma membrane proteins, GPI-APs must be correctly sorted along the different steps of the secretory pathway to their final destination. The presence of both a glycolipid anchor and a protein portion confers special trafficking features to GPI-APs. Here, we discuss the recent advances in the field of GPI-AP trafficking, focusing on the mechanisms regulating their biosynthetic pathway and plasma membrane organization. We also discuss how alterations of these mechanisms can result in different diseases. Finally, we will examine the strict relationship between the trafficking and function of GPI-APs in epithelial cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Behavioral abnormalities in prion protein knockout mice and the potential relevance of PrPc for the cytoskeleton

USDA-ARS?s Scientific Manuscript database

The cellular prion protein (PrPC) is a highly conserved protein, which is anchored to the outer surface of the plasma membrane. Even though its physiological function has already been investigated in different cell or mouse models where PrPC expression is either up-regulated or depleted, its exact p...

Membrane interaction of the N-terminal domain of chemokine receptor CXCR1.

PubMed

Haldar, Sourav; Raghuraman, H; Namani, Trishool; Rajarathnam, Krishna; Chattopadhyay, Amitabha

2010-06-01

The N-terminal domain of chemokine receptors constitutes one of the two critical ligand binding sites, and plays important roles by mediating binding affinity, receptor selectivity, and regulating function. In this work, we monitored the organization and dynamics of a 34-mer peptide of the CXC chemokine receptor 1 (CXCR1) N-terminal domain and its interaction with membranes by utilizing a combination of fluorescence-based approaches and surface pressure measurements. Our results show that the CXCR1 N-domain 34-mer peptide binds vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and upon binding, the tryptophan residues of the peptide experience motional restriction and exhibit red edge excitation shift (REES) of 19nm. These results are further supported by increase in fluorescence anisotropy and mean fluorescence lifetime upon membrane binding. These results constitute one of the first reports demonstrating membrane interaction of the N-terminal domain of CXCR1 and gain relevance in the context of the emerging role of cellular membranes in chemokine signaling.

CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness*

PubMed Central

Grass, G. Daniel; Tolliver, Lauren B.; Bratoeva, Momka; Toole, Bryan P.

2013-01-01

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777–788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer. PMID:23888049

The cystic fibrosis transmembrane conductance regulator (CFTR) and its stability.

PubMed

Meng, Xin; Clews, Jack; Kargas, Vasileios; Wang, Xiaomeng; Ford, Robert C

2017-01-01

The cystic fibrosis transmembrane conductance regulator (CFTR) is responsible for the disease cystic fibrosis (CF). It is a membrane protein belonging to the ABC transporter family functioning as a chloride/anion channel in epithelial cells around the body. There are over 1500 mutations that have been characterised as CF-causing; the most common of these, accounting for ~70 % of CF cases, is the deletion of a phenylalanine at position 508. This leads to instability of the nascent protein and the modified structure is recognised and then degraded by the ER quality control mechanism. However, even pharmacologically 'rescued' F508del CFTR displays instability at the cell's surface, losing its channel function rapidly and it is rapidly removed from the plasma membrane for lysosomal degradation. This review will, therefore, explore the link between stability and structure/function relationships of membrane proteins and CFTR in particular and how approaches to study CFTR structure depend on its stability. We will also review the application of a fluorescence labelling method for the assessment of the thermostability and the tertiary structure of CFTR.

The Min Oscillator Uses MinD-Dependent Conformational Changes in MinE to Spatially Regulate Cytokinesis.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Kyung-Tase; Wu, Wei; Battaile, Kevin P.

In E. coli, MinD recruits MinE to the membrane, leading to a coupled oscillation required for spatial regulation of the cytokinetic Z ring. How these proteins interact, however, is not clear because the MinD-binding regions of MinE are sequestered within a six-stranded {beta} sheet and masked by N-terminal helices. minE mutations that restore interaction between some MinD and MinE mutants were isolated. These mutations alter the MinE structure leading to release of the MinD-binding regions and the N-terminal helices that bind the membrane. Crystallization of MinD-MinE complexes revealed a four-stranded {beta} sheet MinE dimer with the released {beta} strands (MinD-bindingmore » regions) converted to {alpha} helices bound to MinD dimers. These results identify the MinD-dependent conformational changes in MinE that convert it from a latent to an active form and lead to a model of how MinE persists at the MinD-membrane surface.« less

Lipid microdomains and the regulation of ion channel function

PubMed Central

Dart, Caroline

2010-01-01

Many types of ion channel localize to cholesterol and sphingolipid-enriched regions of the plasma membrane known as lipid microdomains or ‘rafts’. The precise physiological role of these unique lipid microenvironments remains elusive due largely to difficulties associated with studying these potentially extremely small and dynamic domains. Nevertheless, increasing evidence suggests that membrane rafts regulate channel function in a number of different ways. Raft-enriched lipids such as cholesterol and sphingolipids exert effects on channel activity either through direct protein–lipid interactions or by influencing the physical properties of the bilayer. Rafts also appear to selectively recruit interacting signalling molecules to generate subcellular compartments that may be important for efficient and selective signal transduction. Direct interaction with raft-associated scaffold proteins such as caveolin can also influence channel function by altering gating kinetics or by affecting trafficking and surface expression. Selective association of ion channels with specific lipid microenvironments within the membrane is thus likely to be an important and fundamental regulatory aspect of channel physiology. This brief review highlights some of the existing evidence for raft modulation of channel function. PMID:20519314

Membrane fouling in a submerged membrane bioreactor: New method and its applications in interfacial interaction quantification.

PubMed

Hong, Huachang; Cai, Xiang; Shen, Liguo; Li, Renjie; Lin, Hongjun

2017-10-01

Quantification of interfacial interactions between two rough surfaces represents one of the most pressing requirements for membrane fouling prediction and control in membrane bioreactors (MBRs). This study firstly constructed regularly rough membrane and particle surfaces by using rigorous mathematical equations. Thereafter, a new method involving surface element integration (SEI) method, differential geometry and composite Simpson's rule was proposed to quantify the interfacial interactions between the two constructed rough surfaces. This new method were then applied to investigate interfacial interactions in a MBR with the data of surface properties of membrane and foulants experimentally measured. The feasibility of the new method was verified. It was found that asperity amplitude and period of the membrane surface exerted profound effects on the total interaction. The new method had broad potential application fields especially including guiding membrane surface design for membrane fouling mitigation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Induction of stable ER–plasma-membrane junctions by Kv2.1 potassium channels

PubMed Central

Fox, Philip D.; Haberkorn, Christopher J.; Akin, Elizabeth J.; Seel, Peter J.; Krapf, Diego; Tamkun, Michael M.

2015-01-01

ABSTRACT Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER–plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K+ channel in the mammalian brain, induces the formation of ER–plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER–plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER–plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca2+ signaling. PMID:25908859

Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission

PubMed Central

Koirala, Sajjan; Guo, Qian; Kalia, Raghav; Bui, Huyen T.; Eckert, Debra M.; Frost, Adam; Shaw, Janet M.

2013-01-01

Mitochondrial fission is mediated by the dynamin-related GTPases Dnm1/Drp1 (yeast/mammals), which form spirals around constricted sites on mitochondria. Additional membrane-associated adaptor proteins (Fis1, Mdv1, Mff, and MiDs) are required to recruit these GTPases from the cytoplasm to the mitochondrial surface. Whether these adaptors participate in both GTPase recruitment and membrane scission is not known. Here we use a yeast strain lacking all fission proteins to identify the minimal combinations of GTPases and adaptors sufficient for mitochondrial fission. Although Fis1 is dispensable for fission, membrane-anchored Mdv1, Mff, or MiDs paired individually with their respective GTPases are sufficient to divide mitochondria. In addition to their role in Drp1 membrane recruitment, MiDs coassemble with Drp1 in vitro. The resulting heteropolymer adopts a dramatically different structure with a narrower diameter than Drp1 homopolymers assembled in isolation. This result demonstrates that an adaptor protein alters the architecture of a mitochondrial dynamin GTPase polymer in a manner that could facilitate membrane constriction and severing activity. PMID:23530241

Extracellular Matrix Signaling from the Cellular Membrane Skeleton to the Nuclear Skeleton: A Model of Gene Regulation

PubMed Central

Lelièvre, Sophie; Weaver, Valerie M.; Bissell, Mina J.

2010-01-01

It is well established that cells must interact with their microenvironment and that such interaction is crucial for coordinated function and homeostasis. However, how cells receive and integrate external signals leading to gene regulation is far from understood. It is now appreciated that two classes of cooperative signals are implicated: a soluble class including hormones and growth factors and a class of insoluble signals emanating from the extracellular matrix (ECM) directly through contact with the cell surface. Using 3-dimensional culture systems and transgenic mice, we have been able to identify some of the elements of this ECM-signaling pathway responsible for gene regulation in rodent mammary gland differentiation and involution. Our major observations are 1) the requirement for a laminin-rich basement membrane; 2) the existence of a cooperative signaling pathway between basement membrane and the lactogenic hormone prolactin (PRL); 3) the importance of β1-integrins and bHLH transcription factor(s) and the presence of DNA response elements (exemplified by BCE-1, located on a milk protein gene, β-casein); and 4) the induction of mammary epithelial cell programmed cell death following degradation of basement membrane. We hypothesize that this cooperative signaling between ECM and PRL may be achieved through integrin- and laminin-directed restructuring of the cytoskeleton leading to profound changes in nuclear architecture and transcription factor localization. We postulate that the latter changes allow the prolactin signal to activate transcription of the β-casein gene. To further understand the molecular mechanisms underlying ECM and hormonal cooperative signaling, we are currently investigating ECM regulation of a “solid-state” signaling pathway including ECM fiber proteins, plasma membrane receptors, cytoskeleton, nuclear matrix and chromatin. We further postulate that disruption of such a pathway may be implicated in cell disorders including transformation and carcinogenesis. PMID:8701089

Surface Properties and Permeability of Poly(Vinylidene Fluoride)-Clays (PVDF/Clays) Composite Membranes

NASA Astrophysics Data System (ADS)

Pramono, E.; Ahdiat, M.; Simamora, A.; Pratiwi, W.; Radiman, C. L.; Wahyuningrum, D.

2017-07-01

Surface properties are important factors that determine the performance of ultrafiltration membranes. This study aimed to investigate the effects of clay addition on the surface properties and membrane permeability of PVDF (poly-vinylidene fluoride) membranes. Three types of clay with different particle size were used in this study, namely montmorillonite-MMT, bentonite-BNT and cloisite 15A-CLS. The PVDF-clay composite membranes were prepared by phase inversion method using PEG as additive. The hydrophobicity of membrane surface was characterized by contact angle. The membrane permeability was determined by dead- end ultrafiltration with a trans-membrane pressure of 2 bars. In contact angle measurement, water contact angle of composite membranes is higher than PVDF membrane. The addition of clays decreased water flux but increased of Dextran rejection. The PVDF-BNT composite membranes reach highest Dextran rejection value of about 93%. The type and particle size of clay affected the hydrophobicity of membrane surface and determined the resulting membrane structure as well as the membrane performance.

An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xiaojun; Department of Biotechnology, Nanchang University, Nanchang, Jiangxi 330031; Chen, Yuan

2014-03-28

Highlights: • Air drying induced the transformation of cell-surface membrane vesicles into pits. • An AFM-based pit-measuring method was developed to measure cell-surface vesicles. • Our method detected at least two populations of cell-surface membrane vesicles. - Abstract: Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM)more » has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.« less

Evaluation of epigallocatechin-3-gallate (EGCG) cross-linked collagen membranes and concerns on osteoblasts.

PubMed

Chu, Chenyu; Deng, Jia; Xiang, Lin; Wu, Yingying; Wei, Xiawei; Qu, Yili; Man, Yi

2016-10-01

Collagen membranes have ideal biological and mechanical properties for supporting infiltration and proliferation of osteoblasts and play a vital role in guided bone regeneration (GBR). However, pure collagen can lead to inflammation, resulting in progressive bone resorption. Therefore, a method for regulating the level of inflammatory cytokines at surgical sites is paramount for the healing process. Epigallocatechin-3-gallate (EGCG) is a component extracted from green tea with numerous biological activities including an anti-inflammatory effect. Herein, we present a novel cross-linked collagen membrane containing different concentrations of EGCG (0.0064%, 0.064%, and 0.64%) to regulate the level of inflammatory factors secreted by pre-osteoblast cells; improve cell proliferation; and increase the tensile strength, wettability, and thermal stability of collagen membranes. Scanning electron microscope images show that the surfaces of collagen membranes became smoother and the collagen fiber diameters became larger with EGCG treatment. Measurement of the water contact angle demonstrated that introducing EGCG improved membrane wettability. Fourier transform infrared spectroscopy analyses indicated that the backbone of collagen was intact, and the thermal stability was significant improved in differential scanning calorimetry. The mechanical properties of 0.064% and 0.64% EGCG-treated collagen membranes were 1.5-fold greater than those of the control. The extent of cross-linking was significantly increased, as determined by a 2,4,6-trinitrobenzenesulfonic acid solution assay. The Cell Counting Kit-8 (CCK-8) and live/dead assays revealed that collagen membrane cross-linked by 0.0064% EGCG induced greater cell proliferation than pure collagen membranes. Additionally, real-time polymerase chain reaction and enzyme-linked immunosorbent assay results showed that EGCG significantly affected the production of inflammatory factors secreted by MC3T3-E1 cells. Taken together, our results indicate that treatment of collagen membranes with appropriate concentrations of EGCG has an anti-inflammatory effect and shows promise for GBR applications. Copyright © 2016. Published by Elsevier B.V.

Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

PubMed

Limsakul, Praopim; Peng, Qin; Wu, Yiqian; Allen, Molly E; Liang, Jing; Remacle, Albert G; Lopez, Tyler; Ge, Xin; Kay, Brian K; Zhao, Huimin; Strongin, Alex Y; Yang, Xiang-Lei; Lu, Shaoying; Wang, Yingxiao

2018-04-19

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Biophysical model of ion transport across human respiratory epithelia allows quantification of ion permeabilities.

PubMed

Garcia, Guilherme J M; Boucher, Richard C; Elston, Timothy C

2013-02-05

Lung health and normal mucus clearance depend on adequate hydration of airway surfaces. Because transepithelial osmotic gradients drive water flows, sufficient hydration of the airway surface liquid depends on a balance between ion secretion and absorption by respiratory epithelia. In vitro experiments using cultures of primary human nasal epithelia and human bronchial epithelia have established many of the biophysical processes involved in airway surface liquid homeostasis. Most experimental studies, however, have focused on the apical membrane, despite the fact that ion transport across respiratory epithelia involves both cellular and paracellular pathways. In fact, the ion permeabilities of the basolateral membrane and paracellular pathway remain largely unknown. Here we use a biophysical model for water and ion transport to quantify ion permeabilities of all pathways (apical, basolateral, paracellular) in human nasal epithelia cultures using experimental (Ussing Chamber and microelectrode) data reported in the literature. We derive analytical formulas for the steady-state short-circuit current and membrane potential, which are for polarized epithelia the equivalent of the Goldman-Hodgkin-Katz equation for single isolated cells. These relations allow parameter estimation to be performed efficiently. By providing a method to quantify all the ion permeabilities of respiratory epithelia, the model may aid us in understanding the physiology that regulates normal airway surface hydration. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides.

PubMed

Saarikangas, Juha; Zhao, Hongxia; Lappalainen, Pekka

2010-01-01

The plasma membrane and the underlying cortical actin cytoskeleton undergo continuous dynamic interplay that is responsible for many essential aspects of cell physiology. Polymerization of actin filaments against cellular membranes provides the force for a number of cellular processes such as migration, morphogenesis, and endocytosis. Plasma membrane phosphoinositides (especially phosphatidylinositol bis- and trisphosphates) play a central role in regulating the organization and dynamics of the actin cytoskeleton by acting as platforms for protein recruitment, by triggering signaling cascades, and by directly regulating the activities of actin-binding proteins. Furthermore, a number of actin-associated proteins, such as BAR domain proteins, are capable of directly deforming phosphoinositide-rich membranes to induce plasma membrane protrusions or invaginations. Recent studies have also provided evidence that the actin cytoskeleton-plasma membrane interactions are misregulated in a number of pathological conditions such as cancer and during pathogen invasion. Here, we summarize the wealth of knowledge on how the cortical actin cytoskeleton is regulated by phosphoinositides during various cell biological processes. We also discuss the mechanisms by which interplay between actin dynamics and certain membrane deforming proteins regulate the morphology of the plasma membrane.

Recycling Endosomes and Viral Infection.

PubMed

Vale-Costa, Sílvia; Amorim, Maria João

2016-03-08

Many viruses exploit specific arms of the endomembrane system. The unique composition of each arm prompts the development of remarkably specific interactions between viruses and sub-organelles. This review focuses on the viral-host interactions occurring on the endocytic recycling compartment (ERC), and mediated by its regulatory Ras-related in brain (Rab) GTPase Rab11. This protein regulates trafficking from the ERC and the trans-Golgi network to the plasma membrane. Such transport comprises intricate networks of proteins/lipids operating sequentially from the membrane of origin up to the cell surface. Rab11 is also emerging as a critical factor in an increasing number of infections by major animal viruses, including pathogens that provoke human disease. Understanding the interplay between the ERC and viruses is a milestone in human health. Rab11 has been associated with several steps of the viral lifecycles by unclear processes that use sophisticated diversified host machinery. For this reason, we first explore the state-of-the-art on processes regulating membrane composition and trafficking. Subsequently, this review outlines viral interactions with the ERC, highlighting current knowledge on viral-host binding partners. Finally, using examples from the few mechanistic studies available we emphasize how ERC functions are adjusted during infection to remodel cytoskeleton dynamics, innate immunity and membrane composition.

Addition of lysophospholipids with large head groups to cells inhibits Shiga toxin binding.

PubMed

Ailte, Ieva; Lingelem, Anne Berit Dyve; Kavaliauskiene, Simona; Bergan, Jonas; Kvalvaag, Audun Sverre; Myrann, Anne-Grethe; Skotland, Tore; Sandvig, Kirsten

2016-07-26

Shiga toxin (Stx), an AB5 toxin, binds specifically to the neutral glycosphingolipid Gb3 at the cell surface before being transported into cells. We here demonstrate that addition of conical lysophospholipids (LPLs) with large head groups inhibit Stx binding to cells whereas LPLs with small head groups do not. Lysophosphatidylinositol (LPI 18:0), the most efficient LPL with the largest head group, was selected for in-depth investigations to study how the binding of Stx is regulated. We show that the inhibition of Stx binding by LPI is reversible and possibly regulated by cholesterol since addition of methyl-β-cyclodextrin (mβCD) reversed the ability of LPI to inhibit binding. LPI-induced inhibition of Stx binding is independent of signalling and membrane turnover as it occurs in fixed cells as well as after depletion of cellular ATP. Furthermore, data obtained with fluorescent membrane dyes suggest that LPI treatment has a direct effect on plasma membrane lipid packing with shift towards a liquid disordered phase in the outer leaflet, while lysophosphoethanolamine (LPE), which has a small head group, does not. In conclusion, our data show that cellular treatment with conical LPLs with large head groups changes intrinsic properties of the plasma membrane and modulates Stx binding to Gb3.

Recycling Endosomes and Viral Infection

PubMed Central

Vale-Costa, Sílvia; Amorim, Maria João

2016-01-01

Many viruses exploit specific arms of the endomembrane system. The unique composition of each arm prompts the development of remarkably specific interactions between viruses and sub-organelles. This review focuses on the viral–host interactions occurring on the endocytic recycling compartment (ERC), and mediated by its regulatory Ras-related in brain (Rab) GTPase Rab11. This protein regulates trafficking from the ERC and the trans-Golgi network to the plasma membrane. Such transport comprises intricate networks of proteins/lipids operating sequentially from the membrane of origin up to the cell surface. Rab11 is also emerging as a critical factor in an increasing number of infections by major animal viruses, including pathogens that provoke human disease. Understanding the interplay between the ERC and viruses is a milestone in human health. Rab11 has been associated with several steps of the viral lifecycles by unclear processes that use sophisticated diversified host machinery. For this reason, we first explore the state-of-the-art on processes regulating membrane composition and trafficking. Subsequently, this review outlines viral interactions with the ERC, highlighting current knowledge on viral-host binding partners. Finally, using examples from the few mechanistic studies available we emphasize how ERC functions are adjusted during infection to remodel cytoskeleton dynamics, innate immunity and membrane composition. PMID:27005655

The membrane as the gatekeeper of infection: Cholesterol in host-pathogen interaction.

PubMed

Kumar, G Aditya; Jafurulla, Md; Chattopadhyay, Amitabha

2016-09-01

The cellular plasma membrane serves as a portal for the entry of intracellular pathogens. An essential step for an intracellular pathogen to gain entry into a host cell therefore is to be able to cross the cell membrane. In this review, we highlight the role of host membrane cholesterol in regulating the entry of intracellular pathogens using insights obtained from work on the interaction of Leishmania and Mycobacterium with host cells. The entry of these pathogens is known to be dependent on host membrane cholesterol. Importantly, pathogen entry is inhibited either upon depletion (or complexation), or enrichment of membrane cholesterol. In other words, an optimum level of host membrane cholesterol is necessary for efficient infection by pathogens. In this overall context, we propose a general mechanism, based on cholesterol-induced conformational changes, involving cholesterol binding sites in host cell surface receptors that are implicated in this process. A therapeutic strategy targeting modulation of membrane cholesterol would have the advantage of avoiding the commonly encountered problem of drug resistance in tackling infection by intracellular pathogens. Insights into the role of host membrane cholesterol in pathogen entry would be instrumental in the development of novel therapeutic strategies to effectively tackle intracellular pathogenesis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Regulated internalization of caveolae

PubMed Central

1994-01-01

Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network. PMID:7962085

PERP, a host tetraspanning membrane protein, is required for S almonella‐induced inflammation

PubMed Central

Hallstrom, Kelly N.; Srikanth, C. V.; Agbor, Terence A.; Dumont, Christopher M.; Peters, Kristen N.; Paraoan, Luminita; Casanova, James E.; Boll, Erik J.

2015-01-01

Summary S almonella enterica  Typhimurium induces intestinal inflammation through the activity of type III secreted effector (T3SE) proteins. Our prior results indicate that the secretion of the T3SE SipA and the ability of SipA to induce epithelial cell responses that lead to induction of polymorphonuclear transepithelial migration are not coupled to its direct delivery into epithelial cells from S almonella. We therefore tested the hypothesis that SipA interacts with a membrane protein located at the apical surface of intestinal epithelial cells. Employing a split ubiquitin yeast‐two‐hybrid screen, we identified the tetraspanning membrane protein, p53 effector related to PMP‐22 (PERP), as a SipA binding partner. SipA and PERP appear to have intersecting activities as we found PERP to be involved in proinflammatory pathways shown to be regulated by SipA. In sum, our studies reveal a critical role for PERP in the pathogenesis of S. Typhimurium, and for the first time demonstrate that SipA, a T3SE protein, can engage a host protein at the epithelial surface. PMID:25486861

Three-Dimensional Geometric Modeling of Membrane-bound Organelles in Ventricular Myocytes: Bridging the Gap between Microscopic Imaging and Mathematical Simulation

PubMed Central

Yu, Zeyun; Holst, Michael J.; Hayashi, Takeharu; Bajaj, Chandrajit L.; Ellisman, Mark H.; McCammon, J. Andrew; Hoshijima, Masahiko

2009-01-01

A general framework of image-based geometric processing is presented to bridge the gap between three-dimensional (3D) imaging that provides structural details of a biological system and mathematical simulation where high-quality surface or volumetric meshes are required. A 3D density map is processed in the order of image pre-processing (contrast enhancement and anisotropic filtering), feature extraction (boundary segmentation and skeletonization), and high-quality and realistic surface (triangular) and volumetric (tetrahedral) mesh generation. While the tool-chain described is applicable to general types of 3D imaging data, the performance is demonstrated specifically on membrane-bound organelles in ventricular myocytes that are imaged and reconstructed with electron microscopic (EM) tomography and two-photon microscopy (T-PM). Of particular interest in this study are two types of membrane-bound Ca2+-handling organelles, namely, transverse tubules (T-tubules) and junctional sarcoplasmic reticulum (jSR), both of which play an important role in regulating the excitation-contraction (E-C) coupling through dynamic Ca2+ mobilization in cardiomyocytes. PMID:18835449

Three-dimensional geometric modeling of membrane-bound organelles in ventricular myocytes: bridging the gap between microscopic imaging and mathematical simulation.

PubMed

Yu, Zeyun; Holst, Michael J; Hayashi, Takeharu; Bajaj, Chandrajit L; Ellisman, Mark H; McCammon, J Andrew; Hoshijima, Masahiko

2008-12-01

A general framework of image-based geometric processing is presented to bridge the gap between three-dimensional (3D) imaging that provides structural details of a biological system and mathematical simulation where high-quality surface or volumetric meshes are required. A 3D density map is processed in the order of image pre-processing (contrast enhancement and anisotropic filtering), feature extraction (boundary segmentation and skeletonization), and high-quality and realistic surface (triangular) and volumetric (tetrahedral) mesh generation. While the tool-chain described is applicable to general types of 3D imaging data, the performance is demonstrated specifically on membrane-bound organelles in ventricular myocytes that are imaged and reconstructed with electron microscopic (EM) tomography and two-photon microscopy (T-PM). Of particular interest in this study are two types of membrane-bound Ca(2+)-handling organelles, namely, transverse tubules (T-tubules) and junctional sarcoplasmic reticulum (jSR), both of which play an important role in regulating the excitation-contraction (E-C) coupling through dynamic Ca(2+) mobilization in cardiomyocytes.

Phosphorylation of actin-binding protein (ABP-280; filamin) by tyrosine kinase p56lck modulates actin filament cross-linking.

PubMed

Pal Sharma, C; Goldmann, Wolfgang H

2004-01-01

Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.

A novel approach for quantitative evaluation of the physicochemical interactions between rough membrane surface and sludge foulants in a submerged membrane bioreactor.

PubMed

Lin, Hongjun; Zhang, Meijia; Mei, Rongwu; Chen, Jianrong; Hong, Huachang

2014-11-01

This study proposed a novel approach for quantitative evaluation of the physicochemical interactions between a particle and rough surface. The approach adopts the composite Simpson's rule to numerically calculate the double integrals in the surface element integration of these physicochemical interactions. The calculation could be achieved by a MATLAB program based on this approach. This approach was then applied to assess the physicochemical interactions between rough membrane surface and sludge foulants in a submerged membrane bioreactor (MBR). The results showed that, as compared with smooth membrane surface, rough membrane surface had a much lower strength of interactions with sludge foulants. Meanwhile, membrane surface morphology significantly affected the strength and properties of the interactions. This study showed that the newly developed approach was feasible, and could serve as a primary tool for investigating membrane fouling in MBRs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Membrane-bound transcription factors: regulated release by RIP or RUP.

PubMed

Hoppe, T; Rape, M; Jentsch, S

2001-06-01

Regulated nuclear transport of transcription factors from cytoplasmic pools is a major route by which eukaryotes control gene expression. Exquisite examples are transcription factors that are kept in a dormant state in the cytosol by membrane anchors; such proteins are released from membranes by proteolytic cleavage, which enables these transcription factors to enter the nucleus. Cleavage can be mediated either by regulated intramembrane proteolysis (RIP) catalysed by specific membrane-bound proteases or by regulated ubiquitin/proteasome-dependent processing (RUP). In both cases processing can be controlled by cues that originate at or in the vicinity of the membrane.

Methods of conditioning direct methanol fuel cells

DOEpatents

Rice, Cynthia; Ren, Xiaoming; Gottesfeld, Shimshon

2005-11-08

Methods for conditioning the membrane electrode assembly of a direct methanol fuel cell ("DMFC") are disclosed. In a first method, an electrical current of polarity opposite to that used in a functioning direct methanol fuel cell is passed through the anode surface of the membrane electrode assembly. In a second method, methanol is supplied to an anode surface of the membrane electrode assembly, allowed to cross over the polymer electrolyte membrane of the membrane electrode assembly to a cathode surface of the membrane electrode assembly, and an electrical current of polarity opposite to that in a functioning direct methanol fuel cell is drawn through the membrane electrode assembly, wherein methanol is oxidized at the cathode surface of the membrane electrode assembly while the catalyst on the anode surface is reduced. Surface oxides on the direct methanol fuel cell anode catalyst of the membrane electrode assembly are thereby reduced.

BTK inhibition results in impaired CXCR4 chemokine receptor surface expression, signaling and function in chronic lymphocytic leukemia.

PubMed

Chen, S-S; Chang, B Y; Chang, S; Tong, T; Ham, S; Sherry, B; Burger, J A; Rai, K R; Chiorazzi, N

2016-04-01

Bruton's tyrosine kinase (BTK) is involved in the regulation of B-cell growth, migration and adhesion. The importance of BTK in cell trafficking is emphasized by the clonal contraction proceeded by lymphocytosis typical for the enzyme inhibitor, ibrutinib, in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Here, we investigated BTK regulation of leukemic B-cell trafficking in a mouse model of aggressive TCL1 CLL-like disease. Inhibiting BTK by ibrutinib reduced surface membrane (sm) levels of CXCR4 but not CXCR5, CD49d and other adhesion/homing receptors. Decreased smCXCR4 levels resulted from blocking receptor signal transduction, which in turn aborted cycling from and to the membrane. This resulted in rapid re-distribution of CLL cells from spleens and lymph nodes into the circulation. CLL cells with impaired smCXCR4 from BTK inhibition failed to home to spleens. These functional changes mainly resulted from inhibition of CXCR4 phosphorylation at Ser339, mediated directly by blocking BTK enzymatic activity and indirectly by affecting the function of downstream targets PLCγ2 and PKCμ, and eventually synthesis of PIM-1 and BTK itself. Our data identify CXCR4 as a key regulator in BTK-mediated CLL-cell retention and have elucidated a complex set of not previously described mechanisms responsible for these effects.

Simple image-based no-wash method for quantitative detection of surface expressed CFTR

PubMed Central

Larsen, Mads Breum; Hu, Jennifer; Frizzell, Raymond A.; Watkins, Simon C.

2016-01-01

Cystic fibrosis (CF) is the most common lethal genetic disease among Caucasians. It is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene, which encodes an apical membrane anion channel that is required for regulating the volume and composition of epithelial secretions. The most common CFTR mutation, present on at least one allele in >90% of CF patients, deletes phenylalanine at position 508 (F508del), which causes the protein to misfold. Endoplasmic reticulum (ER) quality control elicits the degradation of mutant CFTR, compromising its trafficking to the epithelial cell apical membrane. The absence of functional CFTR leads to depletion of airway surface liquid, impaired clearance of mucus and bacteria from the lung, and predisposes to recurrent infections. Ultimately, respiratory failure results from inflammation and bronchiectasis. Although high throughput screening has identified small molecules that can restore the anion transport function of F508del CFTR, they correct less than 15% of WT CFTR activity, yielding insufficient clinical benefit. To date, most primary CF drug discovery assays have employed measurements of CFTR’s anion transport function, a method that depends on the recruitment of a functional CFTR to the cell surface, involves multiple wash steps, and relies on a signal that saturates rapidly. Screening efforts have also included assays for detection of extracellularly HA-tagged or HRP-tagged CFTR, which require multiple washing steps. We have recently developed tools and cell lines that report the correction of mutant CFTR trafficking by currently available small molecules, and have extended this assay to the 96-well format. This new and simple no-wash assay of F508del CFTR at the cell surface may permit the discovery of more efficacious drugs, and hopefully thereby prevent the catastrophic effects of this disease. In addition, the modular design of this platform should make it useful for other diseases where loss-of-function results from folding and/or trafficking defects in membrane proteins. PMID:26361332

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

PubMed Central

Cho, DI; Min, C; Jung, KS; Cheong, SY; Zheng, M; Cheong, SJ; Oak, MH; Cheong, JH; Lee, BK; Kim, KM

2012-01-01

BACKGROUND AND PURPOSE Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH The N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS Shortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONS Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties. PMID:22117524

Surface patterning of polymeric separation membranes and its influence on the filtration performance

NASA Astrophysics Data System (ADS)

Maruf, Sajjad

Polymeric membrane based separation technologies are crucial for addressing the global issues such as water purification. However, continuous operations of these processes are often hindered by fouling which increases mass transport resistance of the membrane to permeation and thus the energy cost, and eventually replacement of the membrane in the system. In comparison to other anti-fouling strategies, the use of controlled surface topography to mitigate fouling has not been realized mainly due to the lack of methods to create targeted topography on the porous membrane surface. This thesis aims to develop a new methodology to create surface-patterned polymeric separation membrane to improve their anti-fouling characteristics during filtration. First, successful fabrication of sub-micron surface patterns directly on a commercial ultrafiltration (UF) membrane surface using nanoimprint lithographic (NIL) technique was demonstrated. Comprehensive filtration studies revealed that the presence of these sub-micron surface patterns mitigates not only the onset of colloidal particle deposition, but also lowers the rate of growth of cake layer after initial deposition, in comparison with un-patterned membranes. The anti-fouling effects were also observed for model protein solutions. Staged filtration experiments, with backwash cleaning, revealed that the permeate flux of the patterned membrane after protein fouling was considerably higher than that of the pristine or un-patterned membrane. In addition to the surface-patterning of UF membranes, successful fabrication of a surface-patterned thin film composite (TFC) membrane was shown for the first time. A two-step fabrication process was carried out by (1) nanoimprinting a polyethersulfone (PES) support using NIL, and (2) forming a thin dense film atop the PES support via interfacial polymerization (IP). Fouling experiments suggest that the surface patterns alter the hydrodynamics at the membrane-feed interface, which is effective in decreasing fouling in dead end filtration system. In summary, this thesis represents the first ever fabrication of functional patterned polymeric separation membrane and systematic investigation of the influence of submicron surface patterns on pressure-driven liquid membrane separations. The results presented here will enable an effective non-chemical surface modification anti-fouling strategy, which can be directly added onto current commercial separation membrane manufacturing route.

Activated release of membrane-anchored TGF-alpha in the absence of cytosol

PubMed Central

1993-01-01

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA- sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol. PMID:8314849

EphA2 cleavage by MT1-MMP triggers single cancer cell invasion via homotypic cell repulsion

PubMed Central

Sugiyama, Nami; Gucciardo, Erika; Tatti, Olga; Varjosalo, Markku; Hyytiäinen, Marko; Gstaiger, Matthias

2013-01-01

Changes in EphA2 signaling can affect cancer cell–cell communication and motility through effects on actomyosin contractility. However, the underlying cell–surface interactions and molecular mechanisms of how EphA2 mediates these effects have remained unclear. We demonstrate here that EphA2 and membrane-anchored membrane type-1 matrix metalloproteinase (MT1-MMP) were selectively up-regulated and coexpressed in invasive breast carcinoma cells, where, upon physical interaction in same cell–surface complexes, MT1-MMP cleaved EphA2 at its Fibronectin type-III domain 1. This cleavage, coupled with EphA2-dependent Src activation, triggered intracellular EphA2 translocation, as well as an increase in RhoA activity and cell junction disassembly, which suggests an overall repulsive effect between cells. Consistent with this, cleavage-prone EphA2-D359I mutant shifted breast carcinoma cell invasion from collective to rounded single-cell invasion within collagen and in vivo. Up-regulated MT1-MMP also codistributed with intracellular EphA2 in invasive cells within human breast carcinomas. These results reveal a new proteolytic regulatory mechanism of cell–cell signaling in cancer invasion. PMID:23629968

The Membrane Mucin Msb2 Regulates Invasive Growth and Plant Infection in Fusarium oxysporum[W

PubMed Central

Pérez-Nadales, Elena; Di Pietro, Antonio

2011-01-01

Fungal pathogenicity in plants requires a conserved mitogen-activated protein kinase (MAPK) cascade homologous to the yeast filamentous growth pathway. How this signaling cascade is activated during infection remains poorly understood. In the soil-borne vascular wilt fungus Fusarium oxysporum, the orthologous MAPK Fmk1 (Fusarium MAPK1) is essential for root penetration and pathogenicity in tomato (Solanum lycopersicum) plants. Here, we show that Msb2, a highly glycosylated transmembrane protein, is required for surface-induced phosphorylation of Fmk1 and contributes to a subset of Fmk1-regulated functions related to invasive growth and virulence. Mutants lacking Msb2 share characteristic phenotypes with the Δfmk1 mutant, including defects in cellophane invasion, penetration of the root surface, and induction of vascular wilt symptoms in tomato plants. In contrast with Δfmk1, Δmsb2 mutants were hypersensitive to cell wall targeting compounds, a phenotype that was exacerbated in a Δmsb2 Δfmk1 double mutant. These results suggest that the membrane mucin Msb2 promotes invasive growth and plant infection upstream of Fmk1 while contributing to cell integrity through a distinct pathway. PMID:21441438

Effect of polymer surface modification on polymer-protein interaction via hydrophilic polymer grafting.

PubMed

Liu, S X; Kim, J-T; Kim, S

2008-04-01

Surface modification of flat sheet ultrafiltration membranes, polyethersulfone (PES), was investigated to improve the hydrophilicity of the membrane surface thereby reducing adsorption of the proteins onto the membrane. Grafting of hydrophilic polymers onto UV/ozone-treated PES was used to improve the hydrophilicity of the commercial PES membranes. Hydrophilic polymers, that is, poly(vinyl alcohol) (PVA), polyethylene glycol (PEG), and chitosan, were employed to graft onto PES membrane surfaces because of their excellent hydrophilic property. The surfaces of modified PES membranes were characterized by contact angle measurement, FTIR, and AFM. The FTIR spectra indicated that PES membranes were successfully modified by grafting of the hydrophilic polymers. The modified PES membranes showed 20% to 50% reduction in contact angle measurements in comparison with those of the virgin PES membrane. The tapping mode AFM technique was employed to investigate the changes of surface topography, cross-section, and root mean square roughness of the modified PES membrane surfaces. The modified PES membranes showed elevated roughness (ranging from 7.0 to 25.7 nm) compared with that of the virgin PES membrane (2.1 nm). It is concluded that grafting of PVA, PEG, or chitosan onto UV/ozone-treated PES membranes increases hydrophilicity and lowers protein adsorption by 20% to 60% compared to the virgin PES membrane. Among the 3 hydrophilic polymers studied, PEG showed the most favorable result in terms of contact angle and protein adsorption.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kubo, Yoshinao; Yoshii, Hiroaki; Kamiyama, Haruka

Ezrin, radixin, and moesin (ERM) proteins supply functional linkage between integral membrane proteins and cytoskeleton in mammalian cells to regulate membrane protein dynamisms and cytoskeleton rearrangement. To assess potential role of the ERM proteins in HIV-1 lifecycle, we examined if suppression of ERM function in human cells expressing HIV-1 infection receptors influences HIV-1 envelope (Env)-mediated HIV-1-vector transduction and cell-cell fusion. Expression of an ezrin dominant negative mutant or knockdown of ezrin, radixin, or moesin with siRNA uniformly decreased transduction titers of HIV-1 vectors having X4-tropic Env. In contrast, transduction titers of R5-tropic Env HIV-1 vectors were decreased only by radixinmore » knockdown: ezrin knockdown had no detectable effects and moesin knockdown rather increased transduction titer. Each of the ERM suppressions had no detectable effects on cell surface expression of CD4, CCR5, and CXCR4 or VSV-Env-mediated HIV-1 vector transductions. Finally, the individual knockdown of ERM mRNAs uniformly decreased efficiency of cell-cell fusion mediated by X4- or R5-tropic Env and HIV-1 infection receptors. These results suggest that (i) the ERM proteins function as positive regulators of infection by X4-tropic HIV-1, (ii) moesin additionally functions as a negative regulator of R5-tropic HIV-1 virus infection at the early step(s) after the membrane fusion, and (iii) receptor protein dynamisms are regulated differently in R5- and X4-tropic HIV-1 infections.« less

Membrane and Integrative Nuclear Fibroblastic Growth Factor Receptor (FGFR) Regulation of FGF-23*

PubMed Central

Han, Xiaobin; Xiao, Zhousheng; Quarles, L. Darryl

2015-01-01

Fibroblastic growth factor receptor 1 (FGFR1) signaling pathways are implicated in the regulation of FGF-23 gene transcription, but the molecular pathways remain poorly defined. We used low molecular weight (LMW, 18 kDa) FGF-2 and high molecular weight (HMW) FGF-2 isoforms, which, respectively, activate cell surface FGF receptors and intranuclear FGFR1, to determine the roles of membrane FGFRs and integrative nuclear FGFR1 signaling (INFS) in the regulation of FGF-23 gene transcription in osteoblasts. We found that LMW-FGF-2 induced NFAT and Ets1 binding to conserved cis-elements in the proximal FGF-23 promoter and stimulated FGF-23 promoter activity through PLCγ/calcineurin/NFAT and MAPK pathways in SaOS-2 and MC3T3-E1 osteoblasts. In contrast, HMW-FGF-2 stimulated FGF-23 promoter activity in osteoblasts through a cAMP-dependent binding of FGFR1 and cAMP-response element-binding protein (CREB) to a conserved cAMP response element (CRE) contiguous with the NFAT binding site in the FGF-23 promoter. Mutagenesis of the NFAT and CRE binding sites, respectively, inhibited the effects of LMW-FGF-2 and HMW-FGF-23 to stimulate FGF-23 promoter activity. FGF-2 activation of both membrane FGFRs and INFS-dependent FGFR1 pathways may provide a means to integrate systemic and local regulation of FGF-23 transcription under diverse physiological and pathological conditions. PMID:25752607

Quantitative membrane proteomics reveals a role for tetraspanin enriched microdomains during entry of human cytomegalovirus

PubMed Central

John, Nessy; Malouli, Daniel

2017-01-01

Human cytomegalovirus (HCMV) depends on and modulates multiple host cell membrane proteins during each stage of the viral life cycle. To gain a global view of the impact of HCMV-infection on membrane proteins, we analyzed HCMV-induced changes in the abundance of membrane proteins in fibroblasts using stable isotope labeling with amino acids (SILAC), membrane fractionation and protein identification by two-dimensional liquid chromatography and tandem mass spectrometry. This systematic approach revealed that CD81, CD44, CD98, caveolin-1 and catenin delta-1 were down-regulated during infection whereas GRP-78 was up-regulated. Since CD81 downregulation was also observed during infection with UV-inactivated virus we hypothesized that this tetraspanin is part of the viral entry process. Interestingly, additional members of the tetraspanin family, CD9 and CD151, were also downregulated during HCMV-entry. Since tetraspanin-enriched microdomains (TEM) cluster host cell membrane proteins including known CMV receptors such as integrins, we studied whether TEMs are required for viral entry. When TEMs were disrupted with the cholesterol chelator methyl-β-cylcodextrin, viral entry was inhibited and this inhibition correlated with reduced surface levels of CD81, CD9 and CD151, whereas integrin levels remained unchanged. Furthermore, simultaneous siRNA-mediated knockdown of multiple tetraspanins inhibited viral entry whereas individual knockdown had little effect suggesting essential, but redundant roles for individual tetraspanins during entry. Taken together, our data suggest that TEM act as platforms for receptors utilized by HCMV for entry into cells. PMID:29121670

Mesoscale studies of ionic closed membranes with polyhedral geometries

DOE PAGES

Olvera de la Cruz, Monica

2016-06-01

Large crystalline molecular shells buckle spontaneously into icosahedra while multicomponent shells buckle into various polyhedra. Continuum elastic theory explains the buckling of closed shells with one elastic component into icosahedra. A generalized elastic model, on the other hand, describes the spontaneous buckling of inhomogeneous shells into regular and irregular polyhedra. By coassembling water-insoluble anionic (–1) amphiphiles with cationic (3+) amphiphiles, we realized ionic vesicles. Results revealed that surface crystalline domains and the unusual shell shapes observed arise from the competition of ionic correlations with charge-regulation. We explain here the mechanism by which these ionic membranes generate a mechanically heterogeneous vesicle.

Fabrication of PAN@TiO2/Ag nanofibrous membrane with high visible light response and satisfactory recyclability for dye photocatalytic degradation

NASA Astrophysics Data System (ADS)

Shi, Yongzheng; Yang, Dongzhi; Li, Yuan; Qu, Jin; Yu, Zhong-Zhen

2017-12-01

Although TiO2-based photocatalysts have exhibited a great potential for degradation of organic pollutants, it is still necessary to simultaneously enhance their visible-light-driven photocatalytic efficiency and physical recyclability. Herein, highly efficient, visible-light-driven photocatalytically active, and recyclable nanofibrous membranes with thin TiO2/Ag heterojunction layer are prepared using electrospun polyacrylonitrile (PAN) nanofibrous membrane as the substrate. By regulating the concentration and hydrolysis process of Ti precursors, TiO2 nanoparticles steadily grow on the PAN nanofibers with high-specific surface area to form a continuous mesoporous shell with the thickness of 20 nm for efficient degradation of organic pollutants. Furthermore, to form a stable heterojunction structure, Ag nanoparticles are deposited on the TiO2 surface by using dopamine as a binder and reductant. The presence of Ag nanoparticles leads to an obvious red-shift from 380 nm to 490 nm, which improves the utilization efficiency of visible light, and reduces the electron/hole recombination rate simultaneously. The resulting PAN@TiO2/Ag membranes hold enhanced photocatalytic activity for methylene blue degradation within 1 h under visible light irradiation, and satisfactory recyclability, which endow them with a great potential for adsorption and photocatalytic applications.

Conformational epitopes at cadherin calcium-binding sites and p120-catenin phosphorylation regulate cell adhesion

PubMed Central

Petrova, Yuliya I.; Spano, MarthaJoy M.; Gumbiner, Barry M.

2012-01-01

We investigated changes in cadherin structure at the cell surface that regulate its adhesive activity. Colo 205 cells are nonadhesive cells with a full but inactive complement of E-cadherin–catenin complexes at the cell surface, but they can be triggered to adhere and form monolayers. We were able to distinguish the inactive and active states of E-cadherin at the cell surface by using a special set of monoclonal antibodies (mAbs). Another set of mAbs binds E-cadherin and strongly activates adhesion. In other epithelial cell types these activating mAbs inhibit growth factor–induced down-regulation of adhesion and epithelial morphogenesis, indicating that these phenomena are also controlled by E-cadherin activity at the cell surface. Both types of mAbs recognize conformational epitopes at different interfaces between extracellular cadherin repeat domains (ECs), especially near calcium-binding sites. Activation also induces p120-catenin dephosphorylation, as well as changes in the cadherin cytoplasmic domain. Moreover, phospho-site mutations indicate that dephosphorylation of specific Ser/Thr residues in the N-terminal domain of p120-catenin mediate adhesion activation. Thus physiological regulation of the adhesive state of E-cadherin involves physical and/or conformational changes in the EC interface regions of the ectodomain at the cell surface that are mediated by catenin-associated changes across the membrane. PMID:22513089

Role of Pgrmc1 in estrogen maintenance of meiotic arrest in zebrafish oocytes through Gper/Egfr.

PubMed

Aizen, Joseph; Thomas, Peter

2015-04-01

The regulation of receptor trafficking to the cell surface and its effect on responses of target cells to growth factors and hormones remain poorly understood. Initial evidence has been recently obtained using cancer cells that surface expression of the epidermal growth factor receptor (EGFR) is dependent on its association with progesterone receptor membrane component 1 (PGRMC1). Estrogen inhibition of oocyte maturation (OM) in zebrafish is mediated through G-protein-coupled estrogen membrane receptor 1 (Gper1) and involves activation of Egfr. Therefore, in this study, the potential roles of Pgrmc1 in the cell surface expression and functions of Egfr in normal cells were investigated in this in vitro OM model of Egfr action using an inhibitor of PGMRC1 signaling, AG205. A single ∼60 kDa protein band, which corresponds to the size of the Pgrmc1 dimer, was detected on plasma membranes of fully grown oocytes by western blotting. Co-treatment with the PGRMC1 inhibitor AG205 (20 μM) blocked the inhibitory effects of 100 nM estradiol-17β and the GPER agonist, G-1, on spontaneous maturation of denuded zebrafish oocytes. Moreover, reversal of these estrogen effects on OM by the EGFR inhibitors AG1478 and AG825 (50 μM) was prevented by co-incubation with the PGRMC1 inhibitor. Inhibition of Pgrmc1 signaling with AG205 also caused a decrease in Egfr-dependent signaling and Egfr expression on oocyte cell membranes. These results indicate that maintenance of Pgrmc1 signaling is required for Egfr expression on zebrafish oocyte cell membranes and for conserving the functions of Egfr in maintaining meiotic arrest through estrogen activation of Gper. © 2015 Society for Endocrinology.

Omniphobic Polyvinylidene Fluoride (PVDF) Membrane for Desalination of Shale Gas Produced Water by Membrane Distillation.

PubMed

Boo, Chanhee; Lee, Jongho; Elimelech, Menachem

2016-11-15

Microporous membranes fabricated from hydrophobic polymers such as polyvinylidene fluoride (PVDF) have been widely used for membrane distillation (MD). However, hydrophobic MD membranes are prone to wetting by low surface tension substances, thereby limiting their use in treating challenging industrial wastewaters, such as shale gas produced water. In this study, we present a facile and scalable approach for the fabrication of omniphobic polyvinylidene fluoride (PVDF) membranes that repel both water and oil. Positive surface charge was imparted to an alkaline-treated PVDF membrane by aminosilane functionalization, which enabled irreversible binding of negatively charged silica nanoparticles (SiNPs) to the membrane through electrostatic attraction. The membrane with grafted SiNPs was then coated with fluoroalkylsilane (perfluorodecyltrichlorosilane) to lower the membrane surface energy. Results from contact angle measurements with mineral oil and surfactant solution demonstrated that overlaying SiNPs with ultralow surface energy significantly enhanced the wetting resistance of the membrane against low surface tension liquids. We also evaluated desalination performance of the modified membrane in direct contact membrane distillation with a synthetic wastewater containing surfactant (sodium dodecyl sulfate) and mineral oil, as well as with shale gas produced water. The omniphobic membrane exhibited a stable MD performance, demonstrating its potential application for desalination of challenging industrial wastewaters containing diverse low surface tension contaminants.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Michael Z.; Simpson, John T.; Aytug, Tolga

Superhydrophobic membrane structures having a beneficial combination of throughput and a selectivity. The membrane structure can include a porous support substrate; and a membrane layer adherently disposed on and in contact with the porous support substrate. The membrane layer can include a nanoporous material having a superhydrophobic surface. The superhydrophobic surface can include a textured surface, and a modifying material disposed on the textured surface. Methods of making and using the membrane structures.

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

PubMed Central

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

1997-01-01

A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ∼40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (∼5–10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ∼3 mM Ca2+ (SW is ∼10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discontinuous surface bilayer by exocytotic fusion events. PMID:9314529

Ras Diffusion Is Sensitive to Plasma Membrane Viscosity

PubMed Central

Goodwin, J. Shawn; Drake, Kimberly R.; Remmert, Catha L.; Kenworthy, Anne K.

2005-01-01

The cell surface contains a variety of barriers and obstacles that slow the lateral diffusion of glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins below the theoretical limit imposed by membrane viscosity. How the diffusion of proteins residing exclusively on the inner leaflet of the plasma membrane is regulated has been largely unexplored. We show here that the diffusion of the small GTPase Ras is sensitive to the viscosity of the plasma membrane. Using confocal fluorescence recovery after photobleaching, we examined the diffusion of green fluorescent protein (GFP)-tagged HRas, NRas, and KRas in COS-7 cells loaded with or depleted of cholesterol, a well-known modulator of membrane bilayer viscosity. In cells loaded with excess cholesterol, the diffusional mobilities of GFP-HRas, GFP-NRas, and GFP-KRas were significantly reduced, paralleling the behavior of the viscosity-sensitive lipid probes DiIC16 and DiIC18. However, the effects of cholesterol depletion on protein and lipid diffusion in cell membranes were highly dependent on the depletion method used. Cholesterol depletion with methyl-β-cyclodextrin slowed Ras diffusion by a viscosity-independent mechanism, whereas overnight cholesterol depletion slightly increased both protein and lipid diffusion. The ability of Ras to sense membrane viscosity may represent a general feature of proteins residing on the cytoplasmic face of the plasma membrane. PMID:15923235

Autophagy-related direct membrane import from ER/cytoplasm into the vacuole or apoplast: a hidden gateway also for secondary metabolites and phytohormones?

PubMed

Kulich, Ivan; Žárský, Viktor

2014-04-29

Transportation of low molecular weight cargoes into the plant vacuole represents an essential plant cell function. Several lines of evidence indicate that autophagy-related direct endoplasmic reticulum (ER) to vacuole (and also, apoplast) transport plays here a more general role than expected. This route is regulated by autophagy proteins, including recently discovered involvement of the exocyst subcomplex. Traffic from ER into the vacuole bypassing Golgi apparatus (GA) acts not only in stress-related cytoplasm recycling or detoxification, but also in developmentally-regulated biopolymer and secondary metabolite import into the vacuole (or apoplast), exemplified by storage proteins and anthocyanins. We propose that this pathway is relevant also for some phytohormones' (e.g., auxin, abscisic acid (ABA) and salicylic acid (SA)) degradation. We hypothesize that SA is not only an autophagy inducer, but also a cargo for autophagy-related ER to vacuole membrane container delivery and catabolism. ER membrane localized enzymes will potentially enhance the area of biosynthetic reactive surfaces, and also, abundant ER localized membrane importers (e.g., ABC transporters) will internalize specific molecular species into the autophagosome biogenesis domain of ER. Such active ER domains may create tubular invaginations of tonoplast into the vacuoles as import intermediates. Packaging of cargos into the ER-derived autophagosome-like containers might be an important mechanism of vacuole and exosome biogenesis and cytoplasm protection against toxic metabolites. A new perspective on metabolic transformations intimately linked to membrane trafficking in plants is emerging.

Autophagy-Related Direct Membrane Import from ER/Cytoplasm into the Vacuole or Apoplast: A Hidden Gateway also for Secondary Metabolites and Phytohormones?

PubMed Central

Kulich, Ivan; Žárský, Viktor

2014-01-01

Transportation of low molecular weight cargoes into the plant vacuole represents an essential plant cell function. Several lines of evidence indicate that autophagy-related direct endoplasmic reticulum (ER) to vacuole (and also, apoplast) transport plays here a more general role than expected. This route is regulated by autophagy proteins, including recently discovered involvement of the exocyst subcomplex. Traffic from ER into the vacuole bypassing Golgi apparatus (GA) acts not only in stress-related cytoplasm recycling or detoxification, but also in developmentally-regulated biopolymer and secondary metabolite import into the vacuole (or apoplast), exemplified by storage proteins and anthocyanins. We propose that this pathway is relevant also for some phytohormones’ (e.g., auxin, abscisic acid (ABA) and salicylic acid (SA)) degradation. We hypothesize that SA is not only an autophagy inducer, but also a cargo for autophagy-related ER to vacuole membrane container delivery and catabolism. ER membrane localized enzymes will potentially enhance the area of biosynthetic reactive surfaces, and also, abundant ER localized membrane importers (e.g., ABC transporters) will internalize specific molecular species into the autophagosome biogenesis domain of ER. Such active ER domains may create tubular invaginations of tonoplast into the vacuoles as import intermediates. Packaging of cargos into the ER-derived autophagosome-like containers might be an important mechanism of vacuole and exosome biogenesis and cytoplasm protection against toxic metabolites. A new perspective on metabolic transformations intimately linked to membrane trafficking in plants is emerging. PMID:24786101

The signaling phospholipid PIP 3 creates a new interaction surface on the nuclear receptor SF-1

DOE PAGES

Blind, Raymond D.; Sablin, Elena P.; Kuchenbecker, Kristopher M.; ...

2014-10-06

We previously reported that lipids PI(4,5)P 2 (PIP 2) and PI(3,4,5)P 3 (PIP 3) bind NR5A nuclear receptors to regulate their activity. Here, the crystal structures of PIP 2 and PIP 3 bound to NR5A1 (SF-1) define a new interaction surface that is organized by the solvent-exposed PIPn headgroups. We find that stabilization by the PIP 3 ligand propagates a signal that increases coactivator recruitment to SF-1, consistent with our earlier work showing that PIP 3 increases SF-1 activity. This newly created surface harbors a cluster of human mutations that lead to endocrine disorders, thus explaining how these puzzling mutationsmore » cripple SF-1 activity. Finally, we propose that this new surface acts as a PIP 3-regulated interface between SF-1 and coregulatory proteins, analogous to the function of membrane-bound phosphoinositides.« less

CURVATURE-DRIVEN MOLECULAR FLOW ON MEMBRANE SURFACE*

PubMed Central

MIKUCKI, MICHAEL; ZHOU, Y. C.

2017-01-01

This work presents a mathematical model for the localization of multiple species of diffusion molecules on membrane surfaces. Morphological change of bilayer membrane in vivo is generally modulated by proteins. Most of these modulations are associated with the localization of related proteins in the crowded lipid environments. We start with the energetic description of the distributions of molecules on curved membrane surface, and define the spontaneous curvature of bilayer membrane as a function of the molecule concentrations on membrane surfaces. A drift-diffusion equation governs the gradient flow of the surface molecule concentrations. We recast the energetic formulation and the related governing equations by using an Eulerian phase field description to define membrane morphology. Computational simulations with the proposed mathematical model and related numerical techniques predict (i) the molecular localization on static membrane surfaces at locations with preferred mean curvatures, and (ii) the generation of preferred mean curvature which in turn drives the molecular localization. PMID:29056778

Versatile antifouling polyethersulfone filtration membranes modified via surface grafting of zwitterionic polymers from a reactive amphiphilic copolymer additive.

PubMed

Zhao, Yi-Fan; Zhang, Pei-Bin; Sun, Jian; Liu, Cui-Jing; Yi, Zhuan; Zhu, Li-Ping; Xu, You-Yi

2015-06-15

Here we describe the development of versatile antifouling polyethersulfone (PES) filtration membranes modified via surface grafting of zwitterionic polymers from a reactive amphiphilic copolymer additive. Amphiphilic polyethersulfone-block-poly(2-hydroxyethyl methacrylate) (PES-b-PHEMA) was beforehand designed and used as the blending additive of PES membranes prepared by phase inversion technique. The surface enriched PHEMA blocks on membrane surface acted as an anchor to immobilize the initiating site. Poly(sulfobetaine methacrylate) (PSBMA) were subsequently grafted onto the PES blend membranes by surface-initiated atom transfer radical polymerization (SI-ATRP). The analysis of surface chemistry confirmed the successful grafting of zwitterionic PSBMA brushes on PES membrane surface. The resulted PES-g-PSBMA membranes were capable of separating proteins from protein solution and oil from oil/water emulsion efficiently. Furthermore, the modified membranes showed high hydrophilicity and strongly antifouling properties due to the incorporation of well-defined PSBMA layer. In addition, the PES-g-PSBMA membranes exhibited excellent blood compatibility and durability during the washing process. The developed antifouling PES membranes are versatile and can find their applications in protein filtration, blood purification and oil/water separation, etc. Copyright © 2015 Elsevier Inc. All rights reserved.

Surface-Enhanced Separation of Water from Hydrocarbons: Potential Dewatering Membranes for the Catalytic Fast Pyrolysis of Pine Biomass

DOE PAGES

Engtrakul, Dr. Chaiwat; Hu, Michael Z.; Bischoff, Brian L; ...

2016-01-01

The impact of surface-selective coatings on water permeation through a membrane when exposed to catalytic fast pyrolysis (CFP) vapor products was studied by tailoring the surface properties of the membrane coating from superhydrophilic to superhydrophobic. Our approach utilized high-performance architectured surface-selective (HiPAS) membranes that were inserted after a CFP reactor. At this insertion point, the inner wall surface of a tubular membrane was exposed to a mixture of water and upgraded product vapors, including light gases and deoxygenated hydrocarbons. Under proper membrane operating conditions, a high selectivity for water over 1-ring upgraded biomass pyrolysis hydrocarbons was observed due to amore » surface-enhanced capillary condensation process. Owing to this surface-enhanced effect, HiPAS membranes have the potential to enable high flux separations suggesting that water can be selectively removed from the CFP product vapors.« less

Surface-Enhanced Separation of Water from Hydrocarbons: Potential Dewatering Membranes for the Catalytic Fast Pyrolysis of Pine Biomass

DOE Office of Scientific and Technical Information (OSTI.GOV)

Engtrakul, Chaiwat; Hu, Michael Z.; Bischoff, Brian L.

2016-10-20

The impact of surface-selective coatings on water permeation through a membrane when exposed to catalytic fast pyrolysis (CFP) vapor products was studied by tailoring the surface properties of the membrane coating from superhydrophilic to superhydrophobic. Our approach used high-performance architectured surface-selective (HiPAS) membranes that were inserted after a CFP reactor. At this insertion point, the inner wall surface of a tubular membrane was exposed to a mixture of water and upgraded product vapors, including light gases and deoxygenated hydrocarbons. Under proper membrane operating conditions, a high selectivity for water over one-ring upgraded biomass pyrolysis hydrocarbons was observed as a resultmore » of a surface-enhanced capillary condensation process. Owing to this surface-enhanced effect, HiPAS membranes have the potential to enable high flux separations, suggesting that water can be selectively removed from the CFP product vapors.« less

Beyond the hydrophobic effect: Critical function of water at biological phase boundaries--A hypothesis.

PubMed

Damodaran, Srinivasan

2015-07-01

Many life-sustaining processes in living cells occur at the membrane-water interface. The pertinent questions that need to be asked are what is the evolutionary reason for biology to choose the membrane-water interface as the site for performing and/or controlling crucial biological reactions and what is the key physical principle that is singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this review, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes and receptor activated processes via manipulating the thermodynamic activity of water at the membrane-water interfacial region. In support of this hypothesis, first we establish that the surface pressure of a lipid monolayer is a direct measure of a reduction in the thermodynamic activity of interfacial water. Second, we show that the surface pressure-dependent activation/inactivation of interfacial enzymes is fundamentally related to their dependence on interfacial water activity. We extend this argument to infer that cells might manipulate activities of membrane-associated biological processes via manipulating the activity of interfacial water via localized compression or expansion of the interface. In this paper, we critically analyze literature data on mechano-activation of large pore ion channels in Escherichia coli spheroplasts and G-proteins in reconstituted lipid vesicles, and show that these pressure-induced activation processes are fundamentally and quantitatively related to changes in the thermodynamic state of interfacial water, caused by mechanical stretching of the bilayer. Copyright © 2015 Elsevier B.V. All rights reserved.

Electrochemical mineral scale prevention and removal on electrically conducting carbon nanotube--polyamide reverse osmosis membranes.

PubMed

Duan, Wenyan; Dudchenko, Alexander; Mende, Elizabeth; Flyer, Celeste; Zhu, Xiaobo; Jassby, David

2014-05-01

The electrochemical prevention and removal of CaSO4 and CaCO3 mineral scales on electrically conducting carbon nanotube - polyamide reverse osmosis membrane was investigated. Different electrical potentials were applied to the membrane surface while filtering model scaling solutions with high saturation indices. Scaling progression was monitored through flux measurements. CaCO3 scale was efficiently removed from the membrane surface through the intermittent application of a 2.5 V potential to the membrane surface, when the membrane acted as an anode. Water oxidation at the anode, which led to proton formation, resulted in the dissolution of deposited CaCO3 crystals. CaSO4 scale formation was significantly retarded through the continuous application of 1.5 V DC to the membrane surface, when the membrane was operated as an anode. The continuous application of a sufficient electrical potential to the membrane surface leads to the formation of a thick layer of counter-ions along the membrane surface that pushed CaSO4 crystal formation away from the membrane surface, allowing the formed crystals to be carried away by the cross-flow. We developed a simple model, based on a modified Poisson-Boltzmann equation, which qualitatively explained our observed experimental results.

Insulin-induced exocytosis regulates the cell surface level of low-density lipoprotein-related protein-1 in Müller Glial cells.

PubMed

Actis Dato, Virginia; Grosso, Rubén A; Sánchez, María C; Fader, Claudio M; Chiabrando, Gustavo A

2018-05-15

Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) is expressed in retinal Müller glial cells (MGCs) and regulates intracellular translocation to the plasma membrane (PM) of the membrane proteins involved in cellular motility and activity. Different functions of MGCs may be influenced by insulin, including the removal of extracellular glutamate in the retina. In the present work, we investigated whether insulin promotes LRP1 translocation to the PM in the Müller glial-derived cell line MIO-M1 (human retinal Müller glial cell-derived cell line). We demonstrated that LRP1 is stored in small vesicles containing an approximate size of 100 nm (mean diameter range of 100-120 nm), which were positive for sortilin and VAMP2, and also incorporated GLUT4 when it was transiently transfected. Next, we observed that LRP1 translocation to the PM was promoted by insulin-regulated exocytosis through intracellular activation of the IR/PI 3 K/Akt axis and Rab-GTPase proteins such as Rab8A and Rab10. In addition, these Rab-GTPases regulated both the constitutive and insulin-induced LRP1 translocation to the PM. Finally, we found that dominant-negative Rab8A and Rab10 mutants impaired insulin-induced intracellular signaling of the IR/PI3K/Akt axis, suggesting that these GTPase proteins as well as the LRP1 level at the cell surface are involved in insulin-induced IR activation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Epidermal growth factor-induced mobilization of a ganglioside-specific sialidase (NEU3) to membrane ruffles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamaguchi, Kazunori; Hata, Keiko; Wada, Tadashi

2006-07-28

Human ganglioside-specific sialidase, NEU3, localized at cell membranes is thought to regulate various biological processes at cell surfaces. We here explored functional subcellular localization of the sialidase by immunofluorescence and found accumulation at leading edges of cell membranes in the presence of serum in culture. In response to EGF, the sialidase redistributed rapidly to ruffling cell membranes of squamous carcinoma A431 cells and co-localized with Rac-1. NEU3 overexpression enhanced Rac-1 activation and cell migration as compared with controls in HeLa cells as well as in A431 cells. Consistent with co-localization with Rac-1 by immunofluorescence, NEU3 was found to co-precipitate withmore » activated Rac bound to GST-PAK-1 fusion protein. NEU3 silencing by siRNA, in contrast, resulted in inhibition of Rac-1 activation. These results indicate that NEU3 is able to mobilize to membrane ruffles in response to growth stimuli and activate the Rac-1 signaling by co-localization with Rac-1, leading to increased cell motility.« less

Membrane Environment Exerts an Important Influence on Rac-Mediated Activation of Phospholipase Cγ2 ▿ †

PubMed Central

Everett, Katy L.; Buehler, Anja; Bunney, Tom D.; Margineanu, Anca; Baxendale, Rhona W.; Vatter, Petra; Retlich, Michael; Walliser, Claudia; Manning, Hugh B.; Neil, Mark A. A.; Dunsby, Christopher; French, Paul M. W.; Gierschik, Peter; Katan, Matilda

2011-01-01

We performed analyses of the molecular mechanisms involved in the regulation of phospholipase Cγ2 (PLCγ2). We identified several regions in the PLCγ-specific array, γSA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLCγ2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLCγ2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLCγ2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation. PMID:21245382

Apical targeting of the formin Diaphanous in Drosophila tubular epithelia

PubMed Central

Rousso, Tal; Shewan, Annette M; Mostov, Keith E; Schejter, Eyal D; Shilo, Ben-Zion

2013-01-01

Apical secretion from epithelial tubes of the Drosophila embryo is mediated by apical F-actin cables generated by the formin-family protein Diaphanous (Dia). Apical localization and activity of Dia are at the core of restricting F-actin formation to the correct membrane domain. Here we identify the mechanisms that target Dia to the apical surface. PI(4,5)P2 levels at the apical membrane regulate Dia localization in both the MDCK cyst model and in Drosophila tubular epithelia. An N-terminal basic domain of Dia is crucial for apical localization, implying direct binding to PI(4,5)P2. Dia apical targeting also depends on binding to Rho1, which is critical for activation-induced conformational change, as well as physically anchoring Dia to the apical membrane. We demonstrate that binding to Rho1 facilitates interaction with PI(4,5)P2 at the plane of the membrane. Together these cues ensure efficient and distinct restriction of Dia to the apical membrane. DOI: http://dx.doi.org/10.7554/eLife.00666.001 PMID:23853710

Acid sphingomyelinase is required for cell surface presentation of Met receptor tyrosine kinase in cancer cells

PubMed Central

Zhu, Linyu; Xiong, Xiahui; Kim, Yongsoon; Okada, Naomi; Lu, Fei; Zhang, Hui

2016-01-01

ABSTRACT Receptor tyrosine kinases (RTKs) are embedded in the lipid bilayer of the plasma membrane, but the specific roles of various lipids in cell signaling remain largely uncharacterized. We have previously found that acid sphingomyelinase (ASM; also known as SMPD1) regulates the conserved DAF-2 (the ortholog IGF-1R in mammals) RTK signaling pathway in Caenorhabditis elegans. How ASM and its catalytic products, ceramides, control RTK signaling pathways remain unclear. Here, we report that ASM regulates the homeostasis of Met, an RTK that is frequently overexpressed in various cancers. Inactivation of ASM led to a rapid loss of Met from the plasma membrane, reduced Met phosphorylation and activation, and induced Met accumulation in the trans-Golgi network (TGN). However, trafficking of integrin β3 and vesicular stomatitis virus glycoprotein (VSVG) was largely unaffected. Knockdown of syntaxin 6 (STX6) also blocked the Golgi exit of Met. Depletion of either ASM or STX6 led to aberrant trafficking of Met to lysosomes, promoting its degradation. Our studies reveal that ASM and ceramides, together with STX6 and cholesterol, constitute a new regulatory mechanism for the exit of Met from the Golgi during its biosynthetic route, which is used to rapidly replenish and regulate the plasma membrane levels of Met in various cancer cells. PMID:27802163

Polarization of IRON-REGULATED TRANSPORTER 1 (IRT1) to the plant-soil interface plays crucial role in metal homeostasis.

PubMed

Barberon, Marie; Dubeaux, Guillaume; Kolb, Cornelia; Isono, Erika; Zelazny, Enric; Vert, Grégory

2014-06-03

In plants, the controlled absorption of soil nutrients by root epidermal cells is critical for growth and development. IRON-REGULATED TRANSPORTER 1 (IRT1) is the main root transporter taking up iron from the soil and is also the main entry route in plants for potentially toxic metals such as manganese, zinc, cobalt, and cadmium. Previous work demonstrated that the IRT1 protein localizes to early endosomes/trans-Golgi network (EE/TGN) and is constitutively endocytosed through a monoubiquitin- and clathrin-dependent mechanism. Here, we show that the availability of secondary non-iron metal substrates of IRT1 (Zn, Mn, and Co) controls the localization of IRT1 between the outer polar domain of the plasma membrane and EE/TGN in root epidermal cells. We also identify FYVE1, a phosphatidylinositol-3-phosphate-binding protein recruited to late endosomes, as an important regulator of IRT1-dependent metal transport and metal homeostasis in plants. FYVE1 controls IRT1 recycling to the plasma membrane and impacts the polar delivery of this transporter to the outer plasma membrane domain. This work establishes a functional link between the dynamics and the lateral polarity of IRT1 and the transport of its substrates, and identifies a molecular mechanism driving polar localization of a cell surface protein in plants.

Surface characterization of dialyzer polymer membranes by imaging ToF-SIMS and quantitative XPS line scans.

PubMed

Holzweber, Markus; Lippitz, Andreas; Krueger, Katharina; Jankowski, Joachim; Unger, Wolfgang E S

2015-03-24

The surfaces of polymeric dialyzer membranes consisting of polysulfone and polyvinylpyrrolidone were investigated regarding the lateral distribution and quantitative surface composition using time-of-flight secondary-ion-mass-spectrometry and x-ray photoelectron spectroscopy. Knowledge of the distribution and composition on the outer surface region is of utmost importance for understanding the biocompatibility of such dialyzer membranes. Both flat membranes and hollow fiber membranes were studied.

Opening up of plasmalemma type-1 VDAC to form apoptotic "find me signal" pathways is essential in early apoptosis - evidence from the pathogenesis of cystic fibrosis resulting from failure of apoptotic cell clearance followed by sterile inflammation.

PubMed

Thinnes, Friedrich P

2014-04-01

Cell membrane-standing type-1 VDAC is involved in cell volume regulation and thus apoptosis. The channel has been shown to figure as a pathway for osmolytes of varying classes, ATP included. An early event in apoptotic cell death is the release of "find me signals" by cells that enter the apoptotic process. ATP is one of those signals. Apoptotic cells this way attract phagocytes for an immunologically silent cell clearance. Thus, whenever apoptosis fails by a blockade of plasmalemma type-1 VDAC processes of sterile inflammation must be assumed for cell elimination. This is evident from a close look on the pathogenetic process of cystic fibrosis (CF). However, in normal airway epithelia two different anion channels cooperate to guarantee an appropriate volume of airway surface liquid (ASL) necessary for surface clearing: the cystic fibrosis conductance regulator (CFTR) and the outwardly rectifying chloride channel (ORCC) complex also called "alternate chloride channel" and under the control of the CFTR. There are arguments, that type-1 VDAC forms the channel part of the ORCC complex, and it has been shown that CFTR and type-1 VDAC co-localize in the apical membranes of human surface respiratory epithelium. In cystic fibrosis, the central cAMP-dependent regulation of ion and water transport via functional CFTR is lost. Here, CFTR molecules do not reach the apical membranes of airway epithelia anymore or work in an insufficient way, respectively. In addition, type-1 VDAC is no longer available to work as a "find me signal" pathway. In consequence, clearing away of apoptotic cells is blocked. There are experimental data on the channel characteristics of type-1 VDAC under the anion channel blocker DIDS (4,4-diisothiocyanato-stilbenedisulphonic acid) that argue in favor of this hypothesis. Together, type-1 VDAC should be kept as a "find me signal" pathway, which may give way to several classes of such signals. Copyright © 2014 Elsevier Inc. All rights reserved.

Surface modification of commercial seawater reverse osmosis membranes by grafting of hydrophilic monomer blended with carboxylated multiwalled carbon nanotubes

NASA Astrophysics Data System (ADS)

Vatanpour, Vahid; Zoqi, Naser

2017-02-01

In this study, modification of commercial seawater reverse osmosis membranes was carried out with simultaneous use of surface grafting and nanoparticle incorporation. Membrane grafting with a hydrophilic acrylic acid monomer and thermal initiator was used to increase membrane surface hydrophilicity. The used nanomaterial was carboxylated multiwalled carbon nanotubes (MWCNTs), which were dispersed in the grafting solution and deposited on membrane surface to reduce fouling by creating polymer brushes and hydrodynamic resistance. Effectiveness of the grafting process (formation of graft layer on membrane surface) was proved by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) analyses. Increase of membrane surface hydrophilicity was approved with contact angle test. First, the grafting was performed on the membrane surfaces with different monomer concentrations, various contact times and several membrane curing times (three variables for optimization). The modified membranes were tested by a cross-flow setup using saline solution for permeability and rejection tests, and bovine serum albumin (BSA) solution for fouling test. The results showed that the modified membranes with 0.75 M of monomer, 3 min contact time and 80 min curing time in an oven at 50 °C presented the highest flux and lowest rejection decline related to the commercial reverse osmosis membrane. In the next step, the optimum grafting condition was selected and the nanotubes with different weight percentages were dispersed in the acrylic acid monomer solution. The membrane containing 0.25 wt% COOH-MWCNTs showed the highest fouling resistance.

Roles of proteolysis in regulation of GPCR function

PubMed Central

Cottrell, GS

2013-01-01

The enzymatic activity of peptidases must be tightly regulated to prevent uncontrolled hydrolysis of peptide bonds, which could have devastating effects on biological systems. Peptidases are often generated as inactive propeptidases, secreted with endogenous inhibitors, or they are compartmentalized. Propeptidases become active after proteolytic removal of N-terminal activation peptides by other peptidases. Some peptidases only become active towards substrates only at certain pHs, thus confining activity to specific compartments or conditions. This review discusses the different roles proteolysis plays in regulating GPCRs. At the cell-surface, certain GPCRs are regulated by the hydrolytic inactivation of bioactive peptides by membrane-anchored peptidases, which prevent signalling. Conversely, cell-surface peptidases can also generate bioactive peptides, which directly activate GPCRs. Alternatively, cell-surface peptidases activated by GPCRs, can generate bioactive peptides to cause transactivation of receptor tyrosine kinases, thereby promoting signalling. Certain peptidases can signal directly to cells, by cleaving GPCR to initiate intracellular signalling cascades. Intracellular peptidases also regulate GPCRs; lysosomal peptidases destroy GPCRs in lysosomes to permanently terminate signalling and mediate down-regulation; endosomal peptidases cleave internalized peptide agonists to regulate GPCR recycling, resensitization and signalling; and soluble intracellular peptidases also participate in GPCR function by regulating the ubiquitination state of GPCRs, thereby altering GPCR signalling and fate. Although the use of peptidase inhibitors has already brought success in the treatment of diseases such as hypertension, the discovery of new regulatory mechanisms involving proteolysis that control GPCRs may provide additional targets to modulate dysregulated GPCR signalling in disease. PMID:23043558

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

PubMed Central

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

2012-01-01

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

Coordinated autoinhibition of F-BAR domain membrane binding and WASp activation by Nervous Wreck.

PubMed

Stanishneva-Konovalova, Tatiana B; Kelley, Charlotte F; Eskin, Tania L; Messelaar, Emily M; Wasserman, Steven A; Sokolova, Olga S; Rodal, Avital A

2016-09-20

Membrane remodeling by Fes/Cip4 homology-Bin/Amphiphysin/Rvs167 (F-BAR) proteins is regulated by autoinhibitory interactions between their SRC homology 3 (SH3) and F-BAR domains. The structural basis of autoregulation, and whether it affects interactions of SH3 domains with other cellular ligands, remain unclear. Here we used single-particle electron microscopy to determine the structure of the F-BAR protein Nervous Wreck (Nwk) in both soluble and membrane-bound states. On membrane binding, Nwk SH3 domains do not completely dissociate from the F-BAR dimer, but instead shift from its concave surface to positions on either side of the dimer. Unexpectedly, along with controlling membrane binding, these autoregulatory interactions inhibit the ability of Nwk-SH3a to activate Wiskott-Aldrich syndrome protein (WASp)/actin related protein (Arp) 2/3-dependent actin filament assembly. In Drosophila neurons, Nwk autoregulation restricts SH3a domain-dependent synaptopod formation, synaptic growth, and actin organization. Our results define structural rearrangements in Nwk that control F-BAR-membrane interactions as well as SH3 domain activities, and suggest that these two functions are tightly coordinated in vitro and in vivo.

Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

PubMed Central

Strasser, Jane E.; Arribas, Monica; Blagoveshchenskaya, Anastasia D.; Cutler, Daniel F.

1999-01-01

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase. PMID:10436017

Enhanced the performance of graphene oxide/polyimide hybrid membrane for CO2 separation by surface modification of graphene oxide using polyethylene glycol

NASA Astrophysics Data System (ADS)

Wu, Li-guang; Yang, Cai-hong; Wang, Ting; Zhang, Xue-yang

2018-05-01

Polyethylene glycol (PEG) with different molecular weights was first used to modify graphene oxide (GO) samples. Subsequently, polyimide (PI) hybrid membranes containing modified-GO were fabricated via in situ polymerization. The separation performance of these hybrid membranes was evaluated using permeation experiments for CO2 and N2 gases. The morphology characterization showed that PEG with suitable molecular weight could be successfully grafted on the GO surface. PEG modification altered the surface properties of GO and introduced defective structures onto GO surface. This caused strong surface polarity and high free volume of membranes containing PEG-modified GO, thereby improving the separation performance of membranes. The addition of PEG-GO with low molecular weight effectively increased gas diffusion through hybrid membranes. The hybrid membranes containing PEG-GO with large molecular weight had high solubility performance for CO2 gas due to the introduction of numerous polar groups into polymeric membranes. With the loading content of modified GO, the CO2 gas permeability of hybrid membranes initially increased but eventually decreased. The optimal content of modified GO in membranes reached 3.0 wt%. When too much PEG added (exceeding 30 g), some impurities formed on GO surface and some aggregates appeared in the resulting hybrid membrane, which depressed the membrane performance.

Hydrophilic, bactericidal nanoheater-enabled reverse osmosis membranes to improve fouling resistance.

PubMed

Ray, Jessica R; Tadepalli, Sirimuvva; Nergiz, Saide Z; Liu, Keng-Ku; You, Le; Tang, Yinjie; Singamaneni, Srikanth; Jun, Young-Shin

2015-06-03

Polyamide (PA) semipermeable membranes typically used for reverse osmosis water treatment processes are prone to fouling, which reduces the amount and quality of water produced. By synergistically coupling the photothermal and bactericidal properties of graphene oxide (GO) nanosheets, gold nanostars (AuNS), and hydrophilic polyethylene glycol (PEG) on PA reverse osmosis membrane surfaces, we have dramatically improved fouling resistance of these membranes. Batch fouling experiments from three classes of fouling are presented: mineral scaling (CaCO3 and CaSO4), organic fouling (humic acid), and biofouling (Escherichia coli). Systematic analyses and a variety of complementary techniques were used to elucidate fouling resistance mechanisms from each layer of modification on the membrane surface. Both mineral scaling and organic fouling were significantly reduced in PA-GO-AuNS-PEG membranes compared to other membranes. The PA-GO-AuNS-PEG membrane was also effective in killing all near-surface bacteria compared to PA membranes. In the PA-GO-AuNS-PEG membrane, the GO nanosheets act as templates for in situ AuNS growth, which then facilitated localized heating upon irradiation by an 808 nm laser inactivating bacteria on the membrane surface. Furthermore, AuNS in the membrane assisted PEG in preventing mineral scaling on the membrane surface. In flow-through flux and foulant rejection tests, PA-GO-AuNS-PEG membranes performed better than PA membranes in the presence of CaSO4 and humic acid model foulants. Therefore, the newly suggested membrane surface modifications will not only reduce fouling from RO feeds, but can improve overall membrane performance. Our innovative membrane design reported in this study can significantly extend the lifetime and water treatment efficacy of reverse osmosis membranes to alleviate escalating global water shortage from rising energy demands.

40 CFR 264.1085 - Standards: Surface impoundments.

Code of Federal Regulations, 2010 CFR

2010-07-01

... the surface impoundment by installing and operating either of the following: (1) A floating membrane... from a surface impoundment using a floating membrane cover shall meet the requirements specified in... floating membrane cover designed to meet the following specifications: (i) The floating membrane cover...

Insulin-Responsive Compartments Containing GLUT4 in 3T3-L1 and CHO Cells: Regulation by Amino Acid Concentrations

PubMed Central

Bogan, Jonathan S.; McKee, Adrienne E.; Lodish, Harvey F.

2001-01-01

In fat and muscle, insulin stimulates glucose uptake by rapidly mobilizing the GLUT4 glucose transporter from a specialized intracellular compartment to the plasma membrane. We describe a method to quantify the relative proportion of GLUT4 at the plasma membrane, using flow cytometry to measure a ratio of fluorescence intensities corresponding to the cell surface and total amounts of a tagged GLUT4 reporter in individual living cells. Using this assay, we demonstrate that both 3T3-L1 and CHO cells contain intracellular compartments from which GLUT4 is rapidly mobilized by insulin and that the initial magnitude and kinetics of redistribution to the plasma membrane are similar in these two cell types when they are cultured identically. Targeting of GLUT4 to a highly insulin-responsive compartment in CHO cells is modulated by culture conditions. In particular, we find that amino acids regulate distribution of GLUT4 to this kinetically defined compartment through a rapamycin-sensitive pathway. Amino acids also modulate the magnitude of insulin-stimulated translocation in 3T3-L1 adipocytes. Our results indicate a novel link between glucose and amino acid metabolism. PMID:11416153

Secretagogue stimulation of neurosecretory cells elicits filopodial extensions uncovering new functional release sites.

PubMed

Papadopulos, Andreas; Martin, Sally; Tomatis, Vanesa M; Gormal, Rachel S; Meunier, Frederic A

2013-12-04

Regulated exocytosis in neurosecretory cells relies on the timely fusion of secretory granules (SGs) with the plasma membrane. Secretagogue stimulation leads to an enlargement of the cell footprint (surface area in contact with the coverslip), an effect previously attributed to exocytic fusion of SGs with the plasma membrane. Using total internal reflection fluorescence microscopy, we reveal the formation of filopodia-like structures in bovine chromaffin and PC12 cells driving the footprint expansion, suggesting the involvement of cortical actin network remodeling in this process. Using exocytosis-incompetent PC12 cells, we demonstrate that footprint enlargement is largely independent of SG fusion, suggesting that vesicular exocytic fusion plays a relatively minor role in filopodial expansion. The footprint periphery, including filopodia, undergoes extensive F-actin remodeling, an effect abolished by the actomyosin inhibitors cytochalasin D and blebbistatin. Imaging of both Lifeact-GFP and the SG marker protein neuropeptide Y-mCherry reveals that SGs actively translocate along newly forming actin tracks before undergoing fusion. Together, these data demonstrate that neurosecretory cells regulate the number of SGs undergoing exocytosis during sustained stimulation by controlling vesicular mobilization and translocation to the plasma membrane through actin remodeling. Such remodeling facilitates the de novo formation of fusion sites.

Leptin-mediated regulation of MT1-MMP localization is KIF1B dependent and enhances gastric cancer cell invasion.

PubMed

Dong, Zhaogang; Xu, Xiaofei; Du, Lutao; Yang, Yongmei; Cheng, Huanhuan; Zhang, Xin; Li, Zewu; Wang, Lili; Li, Juan; Liu, Hui; Qu, Xun; Wang, Chuanxin

2013-05-01

Leptin overexpression is closely correlated with gastric cancer (GC) invasion, but its exact effect and the underlying mechanism in tumorigenesis remain poorly understood. Membrane type 1-matrix metalloproteinase (MT1-MMP), a surface-anchored 'master switch' proteinase, is overexpressed and plays crucial roles in tumor invasion. Here, we characterized the influence of leptin on the generation and surface localization of MT1-MMP in GC and elucidated its molecular mechanisms. Our results revealed that leptin promoted GC cell invasion in vitro by upregulating MT1-MMP expression. Furthermore, cell surface biotinylation assay and flow cytometry demonstrated that the surface expression of MT1-MMP was also enhanced by leptin, and knockdown of kinesin family member 1B (KIF1B, a microtubule plus end-directed monomeric motor protein) by small interference RNA inhibited this process. Notably, coimmunoprecipitation analysis indicated that leptin enhanced the interaction of MT1-MMP with KIF1B in a time-dependent manner, which consequently contributed to GC cell invasion. Moreover, leptin increased MT1-MMP or KIF1B expression by the protein kinase B (AKT) pathway and extracellular signal-regulated kinase 1/2 partially participated in this process. However, only AKT was implicated in the leptin-mediated membrane localization of MT1-MMP. Immunohistochemistry analysis revealed that leptin, MT1-MMP and KIF1B are overexpressed in GC tissues, and they positively correlated with clinical stage and lymph node metastasis. These observations indicate that this regulatory network exists in vivo. Taken together, our findings suggest that leptin is an effective intracellular stimulator of MT1-MMP and that leptin-enhanced cell surface localization of MT1-MMP is dependent on KIF1B, which consequently plays a critical role in GC invasion.

Interactions between Membranes and "Metaphilic" Polypeptide Architectures with Diverse Side-Chain Populations.

PubMed

Lee, Michelle W; Han, Ming; Bossa, Guilherme Volpe; Snell, Carly; Song, Ziyuan; Tang, Haoyu; Yin, Lichen; Cheng, Jianjun; May, Sylvio; Luijten, Erik; Wong, Gerard C L

2017-03-28

At physiological conditions, most proteins or peptides can fold into relatively stable structures that present on their molecular surfaces specific chemical patterns partially smeared out by thermal fluctuations. These nanoscopically defined patterns of charge, hydrogen bonding, and/or hydrophobicity, along with their elasticity and shape stability (folded proteins have Young's moduli of ∼1 × 10 8 Pa), largely determine and limit the interactions of these molecules, such as molecular recognition and allosteric regulation. In this work, we show that the membrane-permeating activity of antimicrobial peptides (AMPs) and cell-penetrating peptides (CPPs) can be significantly enhanced using prototypical peptides with "molten" surfaces: metaphilic peptides with quasi-liquid surfaces and adaptable shapes. These metaphilic peptides have a bottlebrush-like architecture consisting of a rigid helical core decorated with mobile side chains that are terminated by cationic or hydrophobic groups. Computer simulations show that these flexible side chains can undergo significant rearrangement in response to different environments, giving rise to adaptable surface chemistry of the peptide. This quality makes it possible to control their hydrophobicity over a broad range while maintaining water solubility, unlike many AMPs and CPPs. Thus, we are able to show how the activity of these peptides is amplified by hydrophobicity and cationic charge, and rationalize these results using a quantitative mean-field theory. Computer simulations show that the shape-changing properties of the peptides and the resultant adaptive presentation of chemistry play a key enabling role in their interactions with membranes.

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

PubMed Central

Besschetnova, Tatiana Y.; Montefusco, David J.; Asinas, Abdalin E.; Shrout, Anthony L.; Antommattei, Frances M.; Weis, Robert M.

2008-01-01

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. PMID:18711126

How To Functionalize Ceramics by Perfluoroalkylsilanes for Membrane Separation Process? Properties and Application of Hydrophobized Ceramic Membranes.

PubMed

Kujawa, Joanna; Cerneaux, Sophie; Kujawski, Wojciech; Bryjak, Marek; Kujawski, Jan

2016-03-23

The combination of microscopic (atomic force microscopy and scanning electron microscopy) and goniometric (static and dynamic measurements) techniques, and surface characterization (surface free energy determination, critical surface tension, liquid entry pressure, hydraulic permeability) was implemented to discuss the influence of perfluoroalkylsilanes structure and grafting time on the physicochemistry of the created hydrophobic surfaces on the titania ceramic membranes of 5 kD and 300 kD. The impact of molecular structure of perfluoroalkylsilanes modifiers (possessing from 6 to 12 carbon atoms in the fluorinated part of the alkyl chain) and the time of the functionalization process in the range of 5 to 35 h was studied. Based on the scanning electron microscopy with energy-dispersive X-ray spectroscopy, it was found that the localization of grafting molecules depends on the membrane pore size (5 kD or 300 kD). In the case of 5 kD titania membranes, modifiers are attached mainly on the surface and only partially inside the membrane pores, whereas, for 300 kD membranes, the perfluoroalkylsilanes molecules are present within the whole porous structure of the membranes. The application of 4 various types of PFAS molecules enabled for interesting observations and remarks. It was explained how to obtain ceramic membrane surfaces with controlled material (contact angle, roughness, contact angle hysteresis) and separation properties. Highly hydrophobic surfaces with low values of contact angle hysteresis and low roughness were obtained. These surfaces possessed also low values of critical surface tension, which means that surfaces are highly resistant to wetting. This finding is crucial in membrane applicability in separation processes. The obtained and characterized hydrophobic membranes were subsequently applied in air-gap membrane distillation processes. All membranes were very efficient in MD processes, showing good transport and selective properties (∼99% of NaCl salt rejection). Depending on the membrane pore size and used modifiers, the permeate flux was in the range of 0.5-4.5 kg·m(-2)·h(-1) and 0.3-4.2 kg·m(-2)·h(-1) for 5 kD and 300 kD membranes, respectively.

Surface characterization of dialyzer polymer membranes by imaging ToF-SIMS and quantitative XPS line scans

PubMed Central

Holzweber, Markus; Lippitz, Andreas; Krueger, Katharina; Jankowski, Joachim; Unger, Wolfgang E. S.

2015-01-01

The surfaces of polymeric dialyzer membranes consisting of polysulfone and poly-vinylpyrrolidone were investigated regarding the lateral distribution and quantitative surface composition using time-of-flight secondary-ion-mass-spectrometry and x-ray photoelectron spectroscopy. Knowledge of the distribution and composition on the outer surface region is of utmost importance for understanding the biocompatibility of such dialyzer membranes. Both flat membranes and hollow fiber membranes were studied. PMID:25711334

Surface zwitterionicalization of poly(vinylidene fluoride) membranes from the entrapped reactive core-shell silica nanoparticles.

PubMed

Zhu, Li-Jing; Zhu, Li-Ping; Zhang, Pei-Bin; Zhu, Bao-Ku; Xu, You-Yi

2016-04-15

We demonstrate the preparation and properties of poly(vinylidene fluoride) (PVDF) filtration membranes modified via surface zwitterionicalization mediated by reactive core-shell silica nanoparticles (SiO2 NPs). The organic/inorganic hybrid SiO2 NPs grafted with poly(methyl meth acrylate)-block-poly(2-dimethylaminoethyl methacrylate) copolymer (PMMA-b-PDMAEMA) shell were prepared by surface-initiated reversible addition fragmentation chain transfer (SI-RAFT) polymerization and then used as a membrane-making additive of PVDF membranes. The PDMAEMA exposed on membrane surface and pore walls were quaternized into zwitterionic poly(sulfobetaine methacrylate) (PSBMA) using 1,3-propane sultone (1,3-PS) as the quaternization agent. The membrane surface chemistry and morphology were analyzed by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), respectively. The hydrophilicity, permeability and antifouling ability of the investigated membranes were evaluated in detail. It was found that the PSBMA chains brought highly-hydrophilic and strong fouling resistant characteristics to PVDF membranes due to the powerful hydration of zwitterionic surface. The SiO2 cores and PMMA chains in the hybrid NPs play a role of anchors for the linking of PSBMA chains to membrane surface. Compared to the traditional strategies for membrane hydrophilic modification, the developed method in this work combined the advantages of both blending and surface reaction. Copyright © 2016 Elsevier Inc. All rights reserved.

Membrane fouling in a submerged membrane bioreactor: An unified approach to construct topography and to evaluate interaction energy between two randomly rough surfaces.

PubMed

Cai, Xiang; Shen, Liguo; Zhang, Meijia; Chen, Jianrong; Hong, Huachang; Lin, Hongjun

2017-11-01

Quantitatively evaluating interaction energy between two randomly rough surfaces is the prerequisite to quantitatively understand and control membrane fouling in membrane bioreactors (MBRs). In this study, a new unified approach to construct rough topographies and to quantify interaction energy between a randomly rough particle and a randomly rough membrane was proposed. It was found that, natural rough topographies of both foulants and membrane could be well constructed by a modified two-variable Weierstrass-Mandelbrot (WM) function included in fractal theory. Spatial differential relationships between two constructed surfaces were accordingly established. Thereafter, a new approach combining these relationships, surface element integration (SEI) approach and composite Simpson's rule was deduced to calculate the interaction energy between two randomly rough surfaces in a submerged MBR. The obtained results indicate the profound effects of surface morphology on interaction energy and membrane fouling. This study provided a basic approach to investigate membrane fouling and interface behaviors. Copyright © 2017 Elsevier Ltd. All rights reserved.

Direct observation of bacterial deposition onto clean and organic-fouled polyamide membranes.

PubMed

Subramani, Arun; Huang, Xiaofei; Hoek, Eric M V

2009-08-01

Nanofiltration (NF) and reverse osmosis (RO) membranes are commonly applied to produce highly purified water from municipal wastewater effluents. In these applications, biofouling limits overall process performance and increases the cost of operation. Initial bacteria adhesion onto a membrane surface is a critical early step in the overall process of membrane biofouling. However, adsorption of effluent organic matter onto the membrane may precede bacterial deposition and change membrane surface properties. Herein we employed direct microscopic observation to elucidate mechanisms governing bacterial cell deposition onto clean and organic-fouled NF and RO membranes. Bovine serum albumin (BSA) and alginic acid (AA) were used as models for protein and polysaccharide rich organic matter in secondary wastewater effluents. In all experiments, organic fouling increased membrane hydraulic resistance and salt rejection, in addition to interfacial hydrophilicity and roughness. Even though surface hydrophilicity increased, the rougher surfaces presented by organic-fouled membranes produced nano-scale features that promoted localized bacterial deposition. An extended DLVO analysis of bacterial cells and membrane surface properties suggested that bacterial deposition correlated most strongly with the Lewis acid-base free energy of adhesion and root mean square (RMS) roughness, whereas van der Waals and electrostatic free energies were weakly correlated. This was true for both clean and organic-fouled membranes. Bacterial deposition rates were clearly influenced by an antagonistic interplay between macroscopic surface hydrophilicity and nano-scale surface roughness.

Potassium Channels in Regulation of Vascular Smooth Muscle Contraction and Growth

PubMed Central

Jackson, William F.

2017-01-01

Potassium channels importantly contribute to the regulation of vascular smooth muscle (VSM) contraction and growth. They are the dominant ion conductance of the VSM cell membrane and importantly determine and regulate membrane potential. Membrane potential, in turn, regulates the open-state probability of voltage-gated Ca2+ channels (VGCC), Ca2+ influx through VGCC, intracellular Ca2+ and VSM contraction. Membrane potential also affects release of Ca2+ from internal stores and the Ca2+ sensitivity of the contractile machinery such that K+ channels participate in all aspects of regulation of VSM contraction. Potassium channels also regulate proliferation of VSM cells through membrane potential-dependent and membrane potential-independent mechanisms. Vascular smooth muscle cells express multiple isoforms of at least five classes of K+ channels contribute to the regulation of contraction and cell proliferation (growth). This review will examine the structure, expression and function of large-conductance, Ca2+-activated K+ (BKCa) channels, intermediate-conductance Ca2+-activated K+ (KCa3.1) channels, multiple isoforms of voltage-gated K+ (KV) channels, ATP-sensitive K+ (KATP) channels, and inward-rectifier K+ (KIR) channels in both contractile and proliferating VSM cells. PMID:28212804

Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40.

PubMed

Wijesinghe, Kaveesha J; Stahelin, Robert V

2015-12-30

Marburg virus (MARV), which belongs to the virus family Filoviridae, causes hemorrhagic fever in humans and nonhuman primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV carries seven genes, one of which encodes its matrix protein VP40 (mVP40), which regulates the assembly and budding of the virions. Currently, little information is available on mVP40 lipid binding properties. Here, we have investigated the in vitro and cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that is dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid binding interface. Marburg virus (MARV) is a lipid-enveloped filamentous virus from the family Filoviridae. MARV was discovered in 1967, and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40) underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane. In vitro and cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. The results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence, gliding motility and the degradation of complex biopolymers.

PubMed

Veith, Paul D; Glew, Michelle D; Gorasia, Dhana G; Reynolds, Eric C

2017-10-01

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres-Chlorobi-Bacteroidetes superphylum. Proteins secreted by the T9SS have an N-terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C-terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C-terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components. © 2017 John Wiley & Sons Ltd.

Plasmon Resonance Methods in GPCR Signaling and Other Membrane Events

PubMed Central

Alves, I.D.; Park, C.K.; Hruby, V.J.

2005-01-01

The existence of surface guided electromagnetic waves has been theoretically predicted from Maxwell’s equations and investigated during the first decades of the 20th century. However, it is only since the late 1960’s that they have attracted the interest of surface physicists and earned the moniker of “surface plasmon”. With the advent of commercially available instruments and well established theories, the technique has been used to study a wide variety of biochemical and biotechnological phenomena. Spectral response of the resonance condition serves as a sensitive indicator of the optical properties of thin films immobilized within a wavelength of the surface. This enhanced surface sensitivity has provided a boon to the surface sciences, and fosters collaboration between surface chemistry, physics and the ongoing biological and biotechnological revolution. Since then, techniques based on surface plasmons such as Surface Plasmon Resonance (SPR), SPR Imaging, Plasmon Waveguide Resonance (PWR) and others, have been increasingly used to determine the affinity and kinetics of a wide variety of real time molecular interactions such as protein-protein, lipid-protein and ligand-protein, without the need for a molecular tag or label. The physical-chemical methodologies used to immobilize membranes at the surface of these optical devices are reviewed, pointing out advantages and limitations of each method. The paper serves to summarize both historical and more recent developments of these technologies for investigating structure-function aspects of these molecular interactions, and regulation of specific events in signal transduction by G-protein coupled receptors (GPCRs). PMID:16101432

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

Simultaneous bioremediation and biodetection of mercury ion through surface display of carboxylesterase E2 from Pseudomonas aeruginosa PA1.

PubMed

Yin, Kun; Lv, Min; Wang, Qiaoning; Wu, Yixuan; Liao, Chunyang; Zhang, Weiwei; Chen, Lingxin

2016-10-15

Mercury is a toxic heavy metal and presents significant threats to organisms and natural ecosystems. Recently, the mercury remediation as well as its detection by environmental-friendly biotechnology has received increasing attention. In this study, carboxylesterase E2 from mercury-resistant strain Pseudomonas aeruginosa PA1 has been successfully displayed on the outer membrane of Escherichia coli Top10 bacteria to simultaneously adsorb and detect mercury ion (Hg(2+)). The transmission electron microscopy analysis shows that Hg(2+) can be absorbed by carboxylesterase E2 and accumulated on the outer membrane of surface-displayed E. coli bacteria. The adsorption of Hg(2+) followed a physicochemical, equilibrated and saturatable mechanism, which well fits the traditional Langmuir adsorption model. The surface-displayed system can be regenerated through regulating pH values. As its activity can be inhibited by Hg(2+), carboxylesterase E2 has been used to detect the concentration of Hg(2+) in water samples. The developed surface display system will be of great potential in the simultaneous bioremediation and biodetection of environmental mercury pollution. Copyright © 2016 Elsevier Ltd. All rights reserved.

Plasma surface modification of polypropylene track-etched membrane to improve its performance properties

NASA Astrophysics Data System (ADS)

Kravets, L. I.; Elinson, V. M.; Ibragimov, R. G.; Mitu, B.; Dinescu, G.

2018-02-01

The surface and electrochemical properties of polypropylene track-etched membrane treated by plasma of nitrogen, air and oxygen are studied. The effect of the plasma-forming gas composition on the surface morphology is considered. It has been found that the micro-relief of the membrane surface formed under the gas-discharge etching, changes. Moreover, the effect of the non-polymerizing gas plasma leads to formation of oxygen-containing functional groups, mostly carbonyl and carboxyl. It is shown that due to the formation of polar groups on the surface and its higher roughness, the wettability of the plasma-modified membranes improves. In addition, the presence of polar groups on the membrane surface layer modifies its electrochemical properties so that conductivity of plasma-treated membranes increase.

Surface expression of hippocampal NMDA GluN2B receptors regulated by fear conditioning determines its contribution to memory consolidation in adult rats.

PubMed

Sun, Yan-Yan; Cai, Wei; Yu, Jie; Liu, Shu-Su; Zhuo, Min; Li, Bao-Ming; Zhang, Xue-Han

2016-08-04

The number and subtype composition of N-methyl-d-aspartate receptor (NMDAR) at synapses determines their functional properties and role in learning and memory. Genetically increased or decreased amount of GluN2B affects hippocampus-dependent memory in the adult brain. But in some experimental conditions (e.g., memory elicited by a single conditioning trial (1 CS-US)), GluN2B is not a necessary factor, which indicates that the precise role of GluN2B in memory formation requires further exploration. Here, we examined the role of GluN2B in the consolidation of fear memory using two training paradigms. We found that GluN2B was only required for the consolidation of memory elicited by five conditioning trials (5 CS-US), not by 1 CS-US. Strikingly, the expression of membrane GluN2B in CA1was training-strength-dependently increased after conditioning, and that the amount of membrane GluN2B determined its involvement in memory consolidation. Additionally, we demonstrated the increases in the activities of cAMP, ERK, and CREB in the CA1 after conditioning, as well as the enhanced intrinsic excitability and synaptic efficacy in CA1 neurons. Up-regulation of membrane GluN2B contributed to these enhancements. These studies uncover a novel mechanism for the involvement of GluN2B in memory consolidation by its accumulation at the cell surface in response to behavioral training.

The Iron-Responsive Fur/RyhB Regulatory Cascade Modulates the Shigella Outer Membrane Protease IcsP ▿ †

PubMed Central

Africa, Lia A. A.; Murphy, Erin R.; Egan, Nicholas R.; Wigley, Amanda F.; Wing, Helen J.

2011-01-01

Actin-based motility is central to the pathogenicity of the intracellular bacterial pathogen Shigella. Two Shigella outer membrane proteins, IcsA and IcsP, are required for efficient actin-based motility in the host cell cytoplasm, and the genes encoding both proteins are carried on the large virulence plasmid. IcsA triggers actin polymerization on the surface of the bacterium, leading to the formation of an actin tail that allows both intra- and intercellular spread. IcsP, an outer membrane protease, modulates the amount and distribution of the IcsA protein on the bacterial surface through proteolytic cleavage of IcsA. Transcription of icsP is increased in the presence of VirB, a DNA-binding protein that positively regulates many genes carried on the large virulence plasmid. In Shigella dysenteriae, the small regulatory RNA RyhB, which is a member of the iron-responsive Fur regulon, suppresses several virulence-associated phenotypes by downregulating levels of virB in response to iron limitation. Here we show that the Fur/RyhB regulatory pathway downregulates IcsP levels in response to low iron concentrations in Shigella flexneri and that this occurs at the level of transcription through the RyhB-dependent regulation of VirB. These observations demonstrate that in Shigella species the Fur/RyhB regulatory pathway provides a mechanism to finely tune the expression of icsP in response to the low concentrations of free iron predicted to be encountered within colonic epithelial cells. PMID:21859852

Biofouling behavior and performance of forward osmosis membranes with bioinspired surface modification in osmotic membrane bioreactor.

PubMed

Li, Fang; Cheng, Qianxun; Tian, Qing; Yang, Bo; Chen, Qianyuan

2016-07-01

Forward osmosis (FO) has received considerable interest for water and energy related applications in recent years. Biofouling behavior and performance of cellulose triacetate (CTA) forward osmosis membranes with bioinspired surface modification via polydopamine (PD) coating and poly (ethylene glycol) (PEG) grafting (PD-g-PEG) in a submerged osmotic membrane bioreactor (OMBR) were investigated in this work. The modified membranes exhibited lower flux decline than the pristine one in OMBR, confirming that the bioinspired surface modification improved the antifouling ability of the CTA FO membrane. The result showed that the decline of membrane flux related to the increase of the salinity and MLSS concentration of the mixed liquid. It was concluded that the antifouling ability of modified membranes ascribed to the change of surface morphology in addition to the improvement of membrane hydrophilicity. The bioinspired surface modifications might improve the anti-adhesion for the biopolymers and biocake. Copyright © 2016 Elsevier Ltd. All rights reserved.

The effect of polymer surface modification on polymer-protein interaction via interfacial polymerization and hydrophilic polymer grafting

USDA-ARS?s Scientific Manuscript database

Protein membrane separation is prone to fouling on the membrane surface resulting from protein adsorption onto the surface. Surface modification of synthetic membranes is one way to reduce fouling. We investigated surface modification of polyethersulfone (PES) as a way of improving hydrophilicity ...

Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes*

PubMed Central

Park, Dayoung; Brune, Kristin A.; Mitra, Anupam; Marusina, Alina I.; Maverakis, Emanual; Lebrilla, Carlito B.

2015-01-01

Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. PMID:26355101

A nanoparticle-based epigenetic modulator for efficient gene modulation

NASA Astrophysics Data System (ADS)

Pongkulapa, Thanapat

Modulation of gene expression through chromatin remodeling involves epigenetic mechanisms, such as histone acetylation. Acetylation is tightly regulated by two classes of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Molecules that can regulate these enzymes by altering (activating or inhibiting) their functions have become a valuable tool for understanding cell development and diseases. HAT activators, i.e. N-(4-Chloro-(3-trifluoromethyl)phenyl)-2-ethoxybenzamide (CTB), have shown a therapeutic potential for many diseases, including cancer and neurodegeneration. However, these compounds encounter a solubility and a membrane permeability issue, which restricts their full potential for practical usage, especially for in vivo applications. To address this issue, in this work, we developed a nanoparticle-based HAT activator CTB, named Au-CTB, by incorporating a new CTB analogue onto gold nanoparticles (AuNPs) along with a poly(ethylene glycol) moiety and a nuclear localization signal (NLS) peptide to assist with solubility and membrane permeability. We found that our new CTB analogue and Au-CTB could activate HAT activity. Significantly, an increase in potency to activate HAT activity by Au-CTB proved the effectiveness of using the nanoparticle delivery platform. In addition, the versatility of Au-CTB platform permits the attachment of multiple ligands with tunable ratios on the nanoparticle surface via facile surface functionalization of gold nanoparticles. Due to its high delivery efficiency and versatility, Au-CTB can be a powerful platform for applications in epigenetic regulation of gene expression.

Surface modification of poly(vinylidene fluoride) hollow fibre membranes for biogas purification in a gas-liquid membrane contactor system.

PubMed

Jin, Pengrui; Huang, Chuan; Li, Jiaxiang; Shen, Yadong; Wang, Liao

2017-11-01

The wetting of hollow fibre membranes decreases the performance of the liquid-gas membrane contactor for CO 2 capture in biogas upgrading. To solve this problem, in this work, a poly(vinylidene fluoride) (PVDF) hollow fibre membrane for a liquid-gas membrane contactor was coated with a superhydrophobic layer composed of a combination of hydrophobic SiO 2 nanoparticles and polydimethylsiloxane (PDMS) by the method of spray deposition. A rough layer of SiO 2 deposited on the PVDF membrane resulted in an enhanced surface hydrophobicity. The surface structure of the pristine PVDF significantly affected the homogeneity of the generated SiO 2 layer. A uniform surface coating on the PVDF upper layer resulted from the presence of micrometre and nanometre-sized roughness on the surface of the PVDF membrane, which was achieved with a SiO 2 concentration of 4.44 mg ml -1 (0.2 g/45 ml) in the coating solution. As a result, the water contact angle of the modified surface was recorded as 155 ± 3°, which is higher than that of the pristine surface. The high contact angle is advantageous for reducing the wetting of the membrane. Additional mass transfer resistance was introduced by the superhydrophobic layer. In addition, continuous CO 2 absorption tests were carried out in original and modified PVDF hollow fibre membrane contactors, using monoethanolamine (MEA) solution as the absorbent. A long-term stability test revealed that the modified PVDF hollow fibre membrane contactor was able to outperform the original membrane contactor and demonstrated outstanding long-term stability, suggesting that spray deposition is a promising approach to obtain superhydrophobic PVDF membranes for liquid-gas membrane absorption.

Surface modification of poly(vinylidene fluoride) hollow fibre membranes for biogas purification in a gas–liquid membrane contactor system

PubMed Central

Huang, Chuan; Li, Jiaxiang; Shen, Yadong; Wang, Liao

2017-01-01

The wetting of hollow fibre membranes decreases the performance of the liquid–gas membrane contactor for CO2 capture in biogas upgrading. To solve this problem, in this work, a poly(vinylidene fluoride) (PVDF) hollow fibre membrane for a liquid–gas membrane contactor was coated with a superhydrophobic layer composed of a combination of hydrophobic SiO2 nanoparticles and polydimethylsiloxane (PDMS) by the method of spray deposition. A rough layer of SiO2 deposited on the PVDF membrane resulted in an enhanced surface hydrophobicity. The surface structure of the pristine PVDF significantly affected the homogeneity of the generated SiO2 layer. A uniform surface coating on the PVDF upper layer resulted from the presence of micrometre and nanometre-sized roughness on the surface of the PVDF membrane, which was achieved with a SiO2 concentration of 4.44 mg ml−1 (0.2 g/45 ml) in the coating solution. As a result, the water contact angle of the modified surface was recorded as 155 ± 3°, which is higher than that of the pristine surface. The high contact angle is advantageous for reducing the wetting of the membrane. Additional mass transfer resistance was introduced by the superhydrophobic layer. In addition, continuous CO2 absorption tests were carried out in original and modified PVDF hollow fibre membrane contactors, using monoethanolamine (MEA) solution as the absorbent. A long-term stability test revealed that the modified PVDF hollow fibre membrane contactor was able to outperform the original membrane contactor and demonstrated outstanding long-term stability, suggesting that spray deposition is a promising approach to obtain superhydrophobic PVDF membranes for liquid–gas membrane absorption. PMID:29291117

IMPROVING THE QUALITY, AVAILABILITY AND SUSTAINABILITY OF DRINKING WATER SUPPLIES THROUGH ANTIFOULING AND ANTISCALING DESALINATION MEMBRANES

EPA Science Inventory

Surface modification with the selected polymers is expected to reduce the fouling and scaling propensity of desalination membranes by strongly binding water at the membrane surface. Foulants will interact with this bound water layer and not with the membrane surface itself....

Redistribution of caveolae during mitosis

PubMed Central

Boucrot, Emmanuel; Howes, Mark T.; Kirchhausen, Tomas; Parton, Robert G.

2011-01-01

Caveolae form a specialized platform within the plasma membrane that is crucial for an array of important biological functions, ranging from signaling to endocytosis. Using total internal reflection fluorescence (TIRF) and 3D fast spinning-disk confocal imaging to follow caveola dynamics for extended periods, and electron microscopy to obtain high resolution snapshots, we found that the vast majority of caveolae are dynamic with lifetimes ranging from a few seconds to several minutes. Use of these methods revealed a change in the dynamics and localization of caveolae during mitosis. During interphase, the equilibrium between the arrival and departure of caveolae from the cell surface maintains the steady-state distribution of caveolin-1 (Cav1) at the plasma membrane. During mitosis, increased dynamics coupled to an imbalance between the arrival and departure of caveolae from the cell surface induces a redistribution of Cav1 from the plasma membrane to intracellular compartments. These changes are reversed during cytokinesis. The observed redistribution of Cav1 was reproduced by treatment of interphase cells with nocodazole, suggesting that microtubule rearrangements during mitosis can mediate caveolin relocalization. This study provides new insights into the dynamics of caveolae and highlights precise regulation of caveola budding and recycling during mitosis. PMID:21625007

The 78 kDa glucose-regulated protein (GRP78/BIP) is expressed on the cell membrane, is released into cell culture medium and is also present in human peripheral circulation.

PubMed

Delpino, Andrea; Castelli, Mauro

2002-01-01

In human rabdomiosarcoma cells (TE671/RD) chronic exposure to 500 nM thapsigargin (a powerful inhibitor of the endoplasmic reticulum Ca2+-ATPases) resulted in the induction of the stress protein GRP78/BIP. Making use of the surface biotinylation method, followed by the isolation of the GRP78 using ATP-agarose affinity chromatography, it was found that a fraction of the thapsigargin-induced GRP78 is expressed on the cell surface. The presence of GRP78 on the membrane of thapsigargin-treated cells was confirmed by fractionation of cell lysates into a soluble and a membrane fraction, followed by Western blot analysis with an anti-GRP78 antibody. It was also found that conspicuous amounts of GRP78 are present in the culture medium collected from thapsigargin-treated cultures. This extracellular GRP78 originates mostly by an active release from intact cells and does not result solely from the leakage of proteins from dead cells. Moreover, small amounts of circulating, free GRP78 and naturally-occurring anti-GRP78 autoantibodies were detected in the peripheral circulation of healthy human individuals.

Topography of the Dictyostelium discoideum plasma membrane: analysis of membrane asymmetry and intermolecular disulfide bonds.

PubMed

Shiozawa, J A; Jelenska, M M; Jacobson, B S

1987-07-28

Through the application of a unique method for isolating plasma membranes, it was possible to specifically iodinate cytoplasm-exposed plasma membrane proteins in vegetative cells of the cellular slime mold Dictyostelium discoideum. The original procedure [Chaney, L. K., & Jacobson, B. S. (1983) J. Biol. Chem. 258, 10062] which involved coating cells with colloidal silica has been modified to yield a more pure preparation. The presence of the continuous and dense silica pellicle on the outside surface of the isolated plasma membrane permitted the specific labeling of cytoplasm-exposed membrane proteins. Lactoperoxidase-catalyzed iodination was employed to label cell-surface and cytoplasm-exposed membrane proteins. The isolated and radioiodinated membranes were then compared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cell-surface and cytoplasmic face labeling patterns were distinct. A total of 65 proteins were found to be accessible to at least one surface of the membrane. Sixteen intermolecular disulfide bond complexes were observed in the plasma membrane of Dictyostelium; most of these complexes involved glycoproteins and, hence, were exposed to the cell surface.

Design and simulation of the surface shape control system for membrane mirror

NASA Astrophysics Data System (ADS)

Zhang, Gengsheng; Tang, Minxue

2009-11-01

The surface shape control is one of the key technologies for the manufacture of membrane mirror. This paper presents a design of membrane mirror's surface shape control system on the basis of fuzzy logic control. The system contains such function modules as surface shape design, surface shape control, surface shape analysis, and etc. The system functions are realized by using hybrid programming technology of Visual C# and MATLAB. The finite element method is adopted to simulate the surface shape control of membrane mirror. The finite element analysis model is established through ANSYS Parametric Design Language (APDL). ANSYS software kernel is called by the system in background running mode when doing the simulation. The controller is designed by means of controlling the sag of the mirror's central crosssection. The surface shape of the membrane mirror and its optical aberration are obtained by applying Zernike polynomial fitting. The analysis of surface shape control and the simulation of disturbance response are performed for a membrane mirror with 300mm aperture and F/2.7. The result of the simulation shows that by using the designed control system, the RMS wavefront error of the mirror can reach to 142λ (λ=632.8nm), which is consistent to the surface accuracy of the membrane mirror obtained by the large deformation theory of membrane under the same condition.

Dysregulated expression of cell surface glycoprotein CDCP1 in prostate cancer

PubMed Central

Yang, Lifang; Dutta, Sucharita M.; Troyer, Dean A.; Lin, Jefferson B.; Lance, Raymond A.; Nyalwidhe, Julius O.; Drake, Richard R; Semmes, O. John

2015-01-01

CUB-domain-containing protein 1 (CDCP1) is a trans-membrane protein regulator of cell adhesion with a potent pro-migratory function in tumors. Given that proteolytic cleavage of the ectodomain correlates with outside-in oncogenic signaling, we characterized glycosylation in the context of cellular processing and expression of CDCP1 in prostate cancer. We detected 135 kDa full-length and proteolytic processed 70 kDa species in a panel of PCa cell models. The relative expression of full-length CDCP1 correlated with the metastatic potential of syngeneic cell models and an increase in surface membrane expression of CDCP1 was observed in tumor compared to adjacent normal prostate tissues. We demonstrated that glycosylation of CDCP1 is a prerequisite for protein stability and plasma membrane localization, and that the expression level and extent of N-glycosylation of CDCP1 correlated with metastatic status. Interestingly, complex N-linked glycans with sialic acid chains were restricted to the N-terminal half of the ectodomain and absent in the truncated species. Characterization of the extracellular expression of CDCP1 identified novel circulating forms and revealed that extracellular vesicles provide additional processing pathways. Employing immunoaffinity mass spectrometry, we detected elevated levels of circulating CDCP1 in patient urine with high-risk disease. Our results establish that differential glycosylation, cell surface presentation and extracellular expression of CDCP1 are hallmarks of PCa progression. PMID:26497208

An Unconventional Diacylglycerol Kinase That Regulates Phospholipid Synthesis and Nuclear Membrane Growth*♦

PubMed Central

Han, Gil-Soo; O'Hara, Laura; Carman, George M.; Siniossoglou, Symeon

2008-01-01

Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1Δ cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis. PMID:18458075

The plasma membrane as a capacitor for energy and metabolism.

PubMed

Ray, Supriyo; Kassan, Adam; Busija, Anna R; Rangamani, Padmini; Patel, Hemal H

2016-02-01

When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as "capacitors for energy and metabolism." Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell.

Fabrication of high flux and antifouling mixed matrix fumarate-alumoxane/PAN membranes via electrospinning for application in membrane bioreactors

NASA Astrophysics Data System (ADS)

Moradi, Golshan; Zinadini, Sirus; Rajabi, Laleh; Dadari, Soheil

2018-01-01

The nanofibrous Polyacrylonitrile (PAN) membranes embedded with fumarate-alumoxane (Fum-A) nanoparticles were prepared via electrospinning technique as high flux and antifouling membranes for membrane bioreactor (MBR) applications. The effect of Fum-A nanoparticles on membrane morphology, surface hydrophilicity, pure water flux, effluent turbidity and the antifouling property was investigated. Fum-A is a carboxylate-alumoxane nanoparticle covered by extra hydroxyl and carboxylate groups on its surface. By embedding Fum-A nanoparticles into the spinning solution, the surface hydrophilicity and pure water flux of the resulted membranes were improved. The smooth surface of fibers at the low amount of nanoparticles and the agglomeration of nanoparticles at their high concentration were shown in SEM images of the membranes surface. The energy dispersive spectroscopy (EDS) and Fourier transform infrared spectroscopy (FTIR) analysis of the prepared Fum-A/PAN membrane confirmed the presence of carboxylate and hydroxyl functional groups of Fum-A nanoparticles on the surface of the Fum-A nanoparticles containing membrane. The results obtained from the filtration of activated sludge suspension revealed that by addition of a low amount of Fum-A nanoparticles, the irreversible fouling was significantly decreased due to the higher hydrophilicity. The Fum-A/PAN membranes showed superior permeate flux and antifouling properties compared to bare electrospun PAN membrane. Finally, 2 wt.% Fum-A/PAN membrane exhibited the highest FRR of 96% and the lowest irreversible fouling of 4% with excellent durability of antifouling property during twenty repeated activated sludge filtrations.

Involvement of vesicle coat material in casein secretion and surface regeneration

PubMed Central

1976-01-01

The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces. PMID:1254641

Every day I'm rufflin': Calcium sensing and actin dynamics in the growth factor-independent membrane ruffling of professional phagocytes.

PubMed

Schlam, Daniel; Canton, Johnathan

2017-04-03

Professional phagocytes continuously extend dynamic, actin-driven membrane protrusions. These protrusions, often referred to as membrane ruffles, serve a critical role in the essential phagocyte processes of macropinocytosis and phagocytosis. Small GTPases, such as RAC1/2, spatially and temporally regulate membrane ruffle formation. We have recently shown that extracellular calcium regulates the elaboration of membrane ruffles primarily through the synthesis of phosphatidic acid (PtdOH) at the plasma membrane. RAC1/2 guanine nucleotide exchange factors harbouring polybasic stretches are recruited by PtdOH to sites of ruffle formation. Here we discuss our findings and offer perspectives on how the regulation of dynamic actin structures at the plasma membrane by small GTPases is a critical component of phagocyte function.

Glut-1 translocation in FRTL-5 thyroid cells: role of phosphatidylinositol 3-kinase and N-glycosylation.

PubMed

Samih, N; Hovsepian, S; Aouani, A; Lombardo, D; Fayet, G

2000-11-01

It was previously demonstrated that insulin or TSH treatment of FRTL-5 cells resulted in an elevation of glucose transport and in an increase of cell surface expression of the glucose transporter Glut-1. However, the signaling mechanisms leading to the insulin or TSH-induced increase in the cell surface expression of Glut-1 were not investigated. In the present study, we demonstrated that wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3-kinase), interfere both in the signaling pathways of insulin and TSH leading to glucose consumption enhancement and Glut-1 translocation. Two hours after insulin treatment, TSH or cAMP analog (Bu)2cAMP stimulation, glucose transport was increased and most of the intracellular Glut-1 pool was translocated to plasma membranes. Wortmannin or LY294002 blocked the insulin, (Bu)2cAMP, and the TSH-induced translocation of Glut-1. Wortmannin or LY294002 alone did not alter the basal ratio between intracellular and cell surface Glut-1 molecules. These results suggest that in FRTL-5 cells wortmannin and LY294002 inhibited the insulin, (Bu)2cAMP and TSH events leading to Glut-1 translocation from an intracellular compartment to the plasma membrane. Likewise, (Bu)2cAMP effects on glucose transport and Glut-1 translocation to plasma membrane were repressed by PI3-kinase inhibitors but not by the protein kinase A (PKA) inhibitor H89. We suggest that (Bu)2cAMP stimulates Glut-1 translocation to plasma membrane through PI3-kinase-dependent and PKA-independent signaling pathways. To further elucidate mechanisms that regulate the translocation of Glut-1 to cell membrane, we extended this study to the role played by the N-glycosylation in the translocation and in the biological activity of Glut-1 in FRTL-5 cells. For this purpose we used tunicamycin, an inhibitor of the N-glycosylation. Our experiments with tunicamycin clearly showed that both the glycosylated and unglycosylated forms of the transporter reached the cell surface. Likewise, a decrease in glucose consumption (-50%) after treatment of cells with tunicamycin was accompanied by a decrease (-70% vs. control) in the membrane expression of a 50-kDa form of Glut-1 and an increase in its unglycosylated 41-kDa form. These results suggest that carbohydrate moiety is essential for the biological activity of glucose transport but is not required for the translocation of Glut-1 from the intracellular membrane pool to the plasma membrane.

Neuron membrane trafficking and protein kinases involved in autism and ADHD.

PubMed

Kitagishi, Yasuko; Minami, Akari; Nakanishi, Atsuko; Ogura, Yasunori; Matsuda, Satoru

2015-01-30

A brain-enriched multi-domain scaffolding protein, neurobeachin has been identified as a candidate gene for autism patients. Mutations in the synaptic adhesion protein cell adhesion molecule 1 (CADM1) are also associated with autism spectrum disorder, a neurodevelopmental disorder of uncertain molecular origin. Potential roles of neurobeachin and CADM1 have been suggested to a function of vesicle transport in endosomal trafficking. It seems that protein kinase B (AKT) and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) have key roles in the neuron membrane trafficking involved in the pathogenesis of autism. Attention deficit hyperactivity disorder (ADHD) is documented to dopaminergic insufficiencies, which is attributed to synaptic dysfunction of dopamine transporter (DAT). AKT is also essential for the DAT cell-surface redistribution. In the present paper, we summarize and discuss the importance of several protein kinases that regulate the membrane trafficking involved in autism and ADHD, suggesting new targets for therapeutic intervention.

Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

PubMed Central

Cunningham, Orla; Andolfo, Annapaola; Santovito, Maria Lisa; Iuzzolino, Lucia; Blasi, Francesco; Sidenius, Nicolai

2003-01-01

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent-resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA-induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft- associated dimeric uPAR and was completely blocked by cholesterol depletion. PMID:14609946

Thermodynamic assessment of adsorptive fouling with the membranes modified via layer-by-layer self-assembly technique.

PubMed

Shen, Liguo; Cui, Xia; Yu, Genying; Li, Fengquan; Li, Liang; Feng, Shushu; Lin, Hongjun; Chen, Jianrong

2017-05-15

In this study, polyvinylidene fluoride (PVDF) microfiltration membrane was coated by dipping the membrane alternatingly in solutions of the polyelectrolytes (poly-diallyldimethylammonium chloride (PDADMAC) and polystyrenesulfonate (PSS)) via layer-by-layer (LBL) self-assembly technique to improve the membrane antifouling ability. Filtration experiments showed that, sludge cake layer on the coated membrane could be more easily washed off, and moreover, the remained flux ratio (RFR) of the coated membrane was obviously improved as compared with the control membrane. Characterization of the membranes showed that a polyelectrolyte layer was successfully coated on the membrane surfaces, and the hydrophilicity, surface charge and surface morphology of the coated membrane were changed. Based on the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) approaches, quantification of interfacial interactions between foulants and membranes in three different scenarios was achieved. It was revealed that there existed a repulsive energy barrier when a particle foulant adhered to membrane surface, and the enhanced electrostatic double layer (EL) interaction and energy barrier should be responsible for the improved antifouling ability of the coated membrane. This study provided a combined solution to membrane modification and interaction energy evaluation related with membrane fouling simultaneously. Copyright © 2017 Elsevier Inc. All rights reserved.

Composite Membrane with Underwater-Oleophobic Surface for Anti-Oil-Fouling Membrane Distillation.

PubMed

Wang, Zhangxin; Hou, Deyin; Lin, Shihong

2016-04-05

In this study, we fabricated a composite membrane for membrane distillation (MD) by modifying a commercial hydrophobic polyvinylidene fluoride (PVDF) membrane with a nanocomposite coating comprising silica nanoparticles, chitosan hydrogel and fluoro-polymer. The composite membrane exhibits asymmetric wettability, with the modified surface being in-air hydrophilic and underwater oleophobic, and the unmodified surface remaining hydrophobic. By comparing the performance of the composite membrane and the pristine PVDF membrane in direct contact MD experiments using a saline emulsion with 1000 ppm crude oil (in water), we showed that the fabricated composite membrane was significantly more resistant to oil fouling compared to the pristine hydrophobic PVDF membrane. Force spectroscopy was conducted for the interaction between an oil droplet and the membrane surface using a force tensiometer. The difference between the composite membrane and the pristine PVDF membrane in their interaction with an oil droplet served to explain the difference in the fouling propensities between these two membranes observed in MD experiments. The results from this study suggest that underwater oleophobic coating can effectively mitigate oil fouling in MD operations, and that the fabricated composite membrane with asymmetric wettability can enable MD to desalinate hypersaline wastewater with high concentrations of hydrophobic contaminants.

Kidney protection against ischemia/reperfusion injury by myofibrillogenesis regulator-1.

PubMed

Wang, Xiaoreng; Tao, Tianqi; Ding, Rui; Song, Dandan; Liu, Mi; Xie, Yuansheng; Liu, Xiuhua

2014-01-01

Ischemia/reperfusion (I/R) injury is characterized by cytoskeletal reorganization and loss of polarity in proximal tubule epithelial cells. Previously, we showed that myofibrillogenesis regulator (MR)-1 promoted actin organization in cardiomyocytes. MR-1 is also expressed in the kidney. In this study, we investigated MR-1 expression in acute renal failure induced by I/R in Sprague-Dawley rats. We determined the MR-1 expression and the ratio of fibrous actin (F-actin) to globular actin (G-actin). HK-2 cells were treated with or without hypoxia/reoxygenation (H/R), and MR-1 levels were increased by adenoviral overexpression or silenced by RNA interference. I/R and H/R resulted in cellular injury and decreases of MR-1, the F-/G-actin ratio, and myosin light chain (MLC)-2. MR-1 overexpression attenuated H/R-induced cell injury and loss of surface membrane polarity of actin. MR-1 overexpression also increased the expression and phosphorylation of MLC-2 and MLC kinase, which were decreased in MR-1-silenced and H/R-treated cells. Together, these data show that MR-1 promoted actin polarity on the membrane surface and protected HK-2 cells from H/R injury. The mechanism might involve the rapid organization of F-actin through the upregulation and phosphorylation of MLC-2.

Mechanistic insights of intestinal absorption and renal conservation of folate in chronic alcoholism.

PubMed

Wani, Nissar Ahmad; Thakur, Shilpa; Najar, Rauf Ahmad; Nada, Ritambhara; Khanduja, Krishan Lal; Kaur, Jyotdeep

2013-03-01

Folate mediated one-carbon metabolism is of fundamental importance for various cellular processes, including DNA synthesis and methylation of biological molecules. Due to the exogenous requirement of folate in mammals, there exists a well developed epithelial folate transport system for regulation of normal folate homeostasis. The intestinal and renal folate uptake is tightly and diversely regulated and disturbances in folate homeostasis like in alcoholism have pathological consequences. The study was sought to delineate the regulatory mechanism of folate uptake in intestine and reabsorption in renal tubular cells that could evaluate insights of malabsorption during alcoholism. The folate transporters PCFT and RFC were found to be associated with lipid rafts of membrane surfaces in intestine and kidney. Importantly, the observed lower intestinal and renal folate uptake was associated with decreased levels of folate transporter viz. PCFT and RFC in lipid rafts of intestinal and renal membrane surfaces. The decreased association of folate transporters in lipid rafts was associated with decreased protein and mRNA levels. In addition, immunohistochemical studies showed that alcoholic conditions deranged that localization of PCFT and RFC. These findings could explain the possible mechanistic insights that may result in folate malabsorption during alcoholism. Copyright © 2013 Elsevier Inc. All rights reserved.

Key diffusion mechanisms involved in regulating bidirectional water permeation across E. coli outer membrane lectin

PubMed Central

Sachdeva, Shivangi; Kolimi, Narendar; Nair, Sanjana Anilkumar; Rathinavelan, Thenmalarchelvi

2016-01-01

Capsular polysaccharides (CPSs) are major bacterial virulent determinants that facilitate host immune evasion. E. coli group1 K30CPS is noncovalently attached to bacterial surface by Wzi, a lectin. Intriguingly, structure based phylogenetic analysis indicates that Wzi falls into porin superfamily. Molecular dynamics (MD) simulations further shed light on dual role of Wzi as it also functions as a bidirectional passive water specific porin. Such a functional role of Wzi was not realized earlier, due to the occluded pore. While five water specific entry points distributed across extracellular & periplasmic faces regulate the water diffusion involving different mechanisms, a luminal hydrophobic plug governs water permeation across the channel. Coincidently, MD observed open state structure of “YQF” triad is seen in sugar-binding site of sodium-galactose cotransporters, implicating its involvement in K30CPS surface anchorage. Importance of Loop 5 (L5) in membrane insertion is yet another highlight. Change in water diffusion pattern of periplasmic substitution mutants suggests Wzi’s role in osmoregulation by aiding in K30CPS hydration, corroborating earlier functional studies. Water molecules located inside β-barrel of Wzi crystal structure further strengthens the role of Wzi in osmoregulation. Thus, interrupting water diffusion or L5 insertion may reduce bacterial virulence. PMID:27320406

Tunable Membrane Binding of the Intrinsically Disordered Dehydrin Lti30, a Cold-Induced Plant Stress Protein[W

PubMed Central

Eriksson, Sylvia K.; Kutzer, Michael; Procek, Jan; Gröbner, Gerhard; Harryson, Pia

2011-01-01

Dehydrins are intrinsically disordered plant proteins whose expression is upregulated under conditions of desiccation and cold stress. Their molecular function in ensuring plant survival is not yet known, but several studies suggest their involvement in membrane stabilization. The dehydrins are characterized by a broad repertoire of conserved and repetitive sequences, out of which the archetypical K-segment has been implicated in membrane binding. To elucidate the molecular mechanism of these K-segments, we examined the interaction between lipid membranes and a dehydrin with a basic functional sequence composition: Lti30, comprising only K-segments. Our results show that Lti30 interacts electrostatically with vesicles of both zwitterionic (phosphatidyl choline) and negatively charged phospholipids (phosphatidyl glycerol, phosphatidyl serine, and phosphatidic acid) with a stronger binding to membranes with high negative surface potential. The membrane interaction lowers the temperature of the main lipid phase transition, consistent with Lti30’s proposed role in cold tolerance. Moreover, the membrane binding promotes the assembly of lipid vesicles into large and easily distinguishable aggregates. Using these aggregates as binding markers, we identify three factors that regulate the lipid interaction of Lti30 in vitro: (1) a pH dependent His on/off switch, (2) phosphorylation by protein kinase C, and (3) reversal of membrane binding by proteolytic digest. PMID:21665998

Materials and characterization techniques for high-temperature polymer electrolyte membrane fuel cells.

PubMed

Zeis, Roswitha

2015-01-01

The performance of high-temperature polymer electrolyte membrane fuel cells (HT-PEMFC) is critically dependent on the selection of materials and optimization of individual components. A conventional high-temperature membrane electrode assembly (HT-MEA) primarily consists of a polybenzimidazole (PBI)-type membrane containing phosphoric acid and two gas diffusion electrodes (GDE), the anode and the cathode, attached to the two surfaces of the membrane. This review article provides a survey on the materials implemented in state-of-the-art HT-MEAs. These materials must meet extremely demanding requirements because of the severe operating conditions of HT-PEMFCs. They need to be electrochemically and thermally stable in highly acidic environment. The polymer membranes should exhibit high proton conductivity in low-hydration and even anhydrous states. Of special concern for phosphoric-acid-doped PBI-type membranes is the acid loss and management during operation. The slow oxygen reduction reaction in HT-PEMFCs remains a challenge. Phosphoric acid tends to adsorb onto the surface of the platinum catalyst and therefore hampers the reaction kinetics. Additionally, the binder material plays a key role in regulating the hydrophobicity and hydrophilicity of the catalyst layer. Subsequently, the binder controls the electrode-membrane interface that establishes the triple phase boundary between proton conductive electrolyte, electron conductive catalyst, and reactant gases. Moreover, the elevated operating temperatures promote carbon corrosion and therefore degrade the integrity of the catalyst support. These are only some examples how materials properties affect the stability and performance of HT-PEMFCs. For this reason, materials characterization techniques for HT-PEMFCs, either in situ or ex situ, are highly beneficial. Significant progress has recently been made in this field, which enables us to gain a better understanding of underlying processes occurring during fuel cell operation. Various novel tools for characterizing and diagnosing HT-PEMFCs and key components are presented in this review, including FTIR and Raman spectroscopy, confocal Raman microscopy, synchrotron X-ray imaging, X-ray microtomography, and atomic force microscopy.

Materials and characterization techniques for high-temperature polymer electrolyte membrane fuel cells

PubMed Central

2015-01-01

Summary The performance of high-temperature polymer electrolyte membrane fuel cells (HT-PEMFC) is critically dependent on the selection of materials and optimization of individual components. A conventional high-temperature membrane electrode assembly (HT-MEA) primarily consists of a polybenzimidazole (PBI)-type membrane containing phosphoric acid and two gas diffusion electrodes (GDE), the anode and the cathode, attached to the two surfaces of the membrane. This review article provides a survey on the materials implemented in state-of-the-art HT-MEAs. These materials must meet extremely demanding requirements because of the severe operating conditions of HT-PEMFCs. They need to be electrochemically and thermally stable in highly acidic environment. The polymer membranes should exhibit high proton conductivity in low-hydration and even anhydrous states. Of special concern for phosphoric-acid-doped PBI-type membranes is the acid loss and management during operation. The slow oxygen reduction reaction in HT-PEMFCs remains a challenge. Phosphoric acid tends to adsorb onto the surface of the platinum catalyst and therefore hampers the reaction kinetics. Additionally, the binder material plays a key role in regulating the hydrophobicity and hydrophilicity of the catalyst layer. Subsequently, the binder controls the electrode–membrane interface that establishes the triple phase boundary between proton conductive electrolyte, electron conductive catalyst, and reactant gases. Moreover, the elevated operating temperatures promote carbon corrosion and therefore degrade the integrity of the catalyst support. These are only some examples how materials properties affect the stability and performance of HT-PEMFCs. For this reason, materials characterization techniques for HT-PEMFCs, either in situ or ex situ, are highly beneficial. Significant progress has recently been made in this field, which enables us to gain a better understanding of underlying processes occurring during fuel cell operation. Various novel tools for characterizing and diagnosing HT-PEMFCs and key components are presented in this review, including FTIR and Raman spectroscopy, confocal Raman microscopy, synchrotron X-ray imaging, X-ray microtomography, and atomic force microscopy. PMID:25671153

Functional and surface-active membranes from poly(vinylidene fluoride)-graft-poly(acrylic acid) prepared via RAFT-mediated graft copolymerization.

PubMed

Ying, L; Yu, W H; Kang, E T; Neoh, K G

2004-07-06

Poly (vinylidene fluoride) (PVDF) with "living" poly (acrylic acid) (PAAc) side chains (PVDF-g-PAAc) was prepared by reversible addition-fragmentation chain transfer (RAFT)-mediated graft copolymerization of acrylic acid (AAc) with the ozone-pretreated PVDF. The chemical composition and structure of the copolymers were characterized by elemental analysis, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The copolymer could be readily cast into pH-sensitive microfiltration (MF) membranes with enriched living PAAc graft chains on the surface (including the pore surfaces) by phase inversion in an aqueous medium. The surface composition of the membranes was determined by X-ray photoelectron spectroscopy. The morphology of the membranes was characterized by scanning electron microscopy. The pore size distribution of the membranes was found to be much more uniform than that of the corresponding membranes cast from PVDF-g-PAAc prepared by the "conventional" free-radical graft copolymerization process. Most important of all, the MF membranes with surface-tethered PAAc macro chain transfer agents, or the living membrane surfaces, could be further functionalized via surface-initiated block copolymerization with N-isopropylacrylamide (NIPAAM) to obtain the PVDF-g-PAAc-b-PNIPAAM MF membranes, which exhibited both pH- and temperature-dependent permeability to aqueous media.

Electrogenic NBCe1 (SLC4A4), but not electroneutral NBCn1 (SLC4A7), cotransporter undergoes cholinergic-stimulated endocytosis in salivary ParC5 cells.

PubMed

Perry, Clint; Quissell, David O; Reyland, Mary E; Grichtchenko, Irina I

2008-11-01

Cholinergic agonists are major stimuli for fluid secretion in parotid acinar cells. Saliva bicarbonate is essential for maintaining oral health. Electrogenic and electroneutral Na(+)-HCO(3)(-) cotransporters (NBCe1 and NBCn1) are abundant in parotid glands. We previously reported that angiotensin regulates NBCe1 by endocytosis in Xenopus oocytes. Here, we studied cholinergic regulation of NBCe1 and NBCn1 membrane trafficking by confocal fluorescent microscopy and surface biotinylation in parotid epithelial cells. NBCe1 and NBCn1 colocalized with E-cadherin monoclonal antibody at the basolateral membrane (BLM) in polarized ParC5 cells. Inhibition of constitutive recycling with the carboxylic ionophore monensin or the calmodulin antagonist W-13 caused NBCe1 to accumulate in early endosomes with a parallel loss from the BLM, suggesting that NBCe1 is constitutively endocytosed. Carbachol and PMA likewise caused redistribution of NBCe1 from BLM to early endosomes. The PKC inhibitor, GF-109203X, blocked this redistribution, indicating a role for PKC. In contrast, BLM NBCn1 was not downregulated in parotid acinar cells treated with constitutive recycling inhibitors, cholinergic stimulators, or PMA. We likewise demonstrate striking differences in regulation of membrane trafficking of NBCe1 vs. NBCn1 in resting and stimulated cells. We speculate that endocytosis of NBCe1, which coincides with the transition to a steady-state phase of stimulated fluid secretion, could be a part of acinar cell adjustment to a continuous secretory response. Stable association of NBCn1 at the membrane may facilitate constitutive uptake of HCO(3)(-) across the BLM, thus supporting HCO(3)(-) luminal secretion and/or maintaining acid-base homeostasis in stimulated cells.

Fatty acids from membrane lipids become incorporated into lipid bodies during Myxococcus xanthus differentiation.

PubMed

Bhat, Swapna; Boynton, Tye O; Pham, Dan; Shimkets, Lawrence J

2014-01-01

Myxococcus xanthus responds to amino acid limitation by producing fruiting bodies containing dormant spores. During development, cells produce triacylglycerides in lipid bodies that become consumed during spore maturation. As the cells are starved to induce development, the production of triglycerides represents a counterintuitive metabolic switch. In this paper, lipid bodies were quantified in wild-type strain DK1622 and 33 developmental mutants at the cellular level by measuring the cross sectional area of the cell stained with the lipophilic dye Nile red. We provide five lines of evidence that triacylglycerides are derived from membrane phospholipids as cells shorten in length and then differentiate into myxospores. First, in wild type cells, lipid bodies appear early in development and their size increases concurrent with an 87% decline in membrane surface area. Second, developmental mutants blocked at different stages of shortening and differentiation accumulated lipid bodies proportionate with their cell length with a Pearson's correlation coefficient of 0.76. Third, peripheral rods, developing cells that do not produce lipid bodies, fail to shorten. Fourth, genes for fatty acid synthesis are down-regulated while genes for fatty acid degradation are up regulated. Finally, direct movement of fatty acids from membrane lipids in growing cells to lipid bodies in developing cells was observed by pulse labeling cells with palmitate. Recycling of lipids released by Programmed Cell Death appears not to be necessary for lipid body production as a fadL mutant was defective in fatty acid uptake but proficient in lipid body production. The lipid body regulon involves many developmental genes that are not specifically involved in fatty acid synthesis or degradation. MazF RNA interferase and its target, enhancer-binding protein Nla6, appear to negatively regulate cell shortening and TAG accumulation whereas most cell-cell signals activate these processes.

Confined diffusion of transmembrane proteins and lipids induced by the same actin meshwork lining the plasma membrane.

PubMed

Fujiwara, Takahiro K; Iwasawa, Kokoro; Kalay, Ziya; Tsunoyama, Taka A; Watanabe, Yusuke; Umemura, Yasuhiro M; Murakoshi, Hideji; Suzuki, Kenichi G N; Nemoto, Yuri L; Morone, Nobuhiro; Kusumi, Akihiro

2016-04-01

The mechanisms by which the diffusion rate in the plasma membrane (PM) is regulated remain unresolved, despite their importance in spatially regulating the reaction rates in the PM. Proposed models include entrapment in nanoscale noncontiguous domains found in PtK2 cells, slow diffusion due to crowding, and actin-induced compartmentalization. Here, by applying single-particle tracking at high time resolutions, mainly to the PtK2-cell PM, we found confined diffusion plus hop movements (termed "hop diffusion") for both a nonraft phospholipid and a transmembrane protein, transferrin receptor, and equal compartment sizes for these two molecules in all five of the cell lines used here (actual sizes were cell dependent), even after treatment with actin-modulating drugs. The cross-section size and the cytoplasmic domain size both affected the hop frequency. Electron tomography identified the actin-based membrane skeleton (MSK) located within 8.8 nm from the PM cytoplasmic surface of PtK2 cells and demonstrated that the MSK mesh size was the same as the compartment size for PM molecular diffusion. The extracellular matrix and extracellular domains of membrane proteins were not involved in hop diffusion. These results support a model of anchored TM-protein pickets lining actin-based MSK as a major mechanism for regulating diffusion. © 2016 Fujiwara et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The attenuated inflammation of MPL is due to the lack of CD14-dependent tight dimerization of the TLR4/MD2 complex at the plasma membrane.

PubMed

Tanimura, Natsuko; Saitoh, Shin-Ichiroh; Ohto, Umeharu; Akashi-Takamura, Sachiko; Fujimoto, Yukari; Fukase, Koichi; Shimizu, Toshiyuki; Miyake, Kensuke

2014-06-01

TLR4/MD-2 senses lipid A, activating the MyD88-signaling pathway on the plasma membrane and the TRIF-signaling pathway after CD14-mediated TLR4/MD-2 internalization into endosomes. Monophosphoryl lipid A (MPL), a detoxified derivative of lipid A, is weaker than lipid A in activating the MyD88-dependent pathway. Little is known, however, about mechanisms underlying the attenuated activation of MyD88-dependent pathways. We here show that MPL was impaired in induction of CD14-dependent TLR4/MD-2 dimerization compared with lipid A. Impaired TLR4/MD-2 dimerization decreased CD14-mediated TNFα production. In contrast, MPL was comparable to lipid A in CD14-independent MyD88-dependent TNFα production and TRIF-dependent responses including cell surface CD86 up-regulation and IFNβ induction. Although CD86 up-regulation is dependent on TRIF signaling, it was induced by TLR4/MD-2 at the plasma membrane. These results revealed that the attenuated MPL responses were due to CD14-initiated responses at the plasma membrane, but not just to responses initiated by MyD88, that is, MPL was specifically unable to induce CD14-dependent TLR4/MD-2 dimerization that selectively enhances MyD88-mediated responses at the plasma membrane. © The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Hierarchical Composite Membranes with Robust Omniphobic Surface Using Layer-By-Layer Assembly Technique.

PubMed

Woo, Yun Chul; Kim, Youngjin; Yao, Minwei; Tijing, Leonard D; Choi, June-Seok; Lee, Sangho; Kim, Seung-Hyun; Shon, Ho Kyong

2018-02-20

In this study, composite membranes were fabricated via layer-by-layer (LBL) assembly of negatively charged silica aerogel (SiA) and 1H,1H,2H,2H-perfluorodecyltriethoxysilane (FTCS) on a polyvinylidene fluoride phase inversion membrane and interconnecting them with positively charged poly(diallyldimethylammonium chloride) (PDDA) via electrostatic interaction. The results showed that the PDDA-SiA-FTCS coated membrane had significantly enhanced the membrane structure and properties. New trifluoromethyl and tetrafluoroethylene bonds appeared at the surface of the coated membrane, which led to lower surface free energy of the composite membrane. Additionally, the LBL membrane showed increased surface roughness. The improved structure and property gave the LBL membrane an omniphobic property, as indicated by its good wetting resistance. The membrane performed a stable air gap membrane distillation (AGMD) flux of 11.22 L/m 2 h with very high salt rejection using reverse osmosis brine from coal seam gas produced water as feed with the addition of up to 0.5 mM SDS solution. This performance was much better compared to those of the neat membrane. The present study suggests that the enhanced membrane properties with good omniphobicity via LBL assembly make the porous membranes suitable for long-term AGMD operation with stable permeation flux when treating challenging saline wastewater containing low surface tension organic contaminants.

An analysis of the relationships between subthreshold electrical properties and excitability in skeletal muscle

PubMed Central

L.-H. Huang, Christopher; Fraser, James A.

2011-01-01

Skeletal muscle activation requires action potential (AP) initiation followed by its sarcolemmal propagation and tubular excitation to trigger Ca2+ release and contraction. Recent studies demonstrate that ion channels underlying the resting membrane conductance (GM) of fast-twitch mammalian muscle fibers are highly regulated during muscle activity. Thus, onset of activity reduces GM, whereas prolonged activity can markedly elevate GM. Although these observations implicate GM regulation in control of muscle excitability, classical theoretical studies in un-myelinated axons predict little influence of GM on membrane excitability. However, surface membrane morphologies differ markedly between un-myelinated axons and muscle fibers, predominantly because of the tubular (t)-system of muscle fibers. This study develops a linear circuit model of mammalian muscle fiber and uses this to assess the role of subthreshold electrical properties, including GM changes during muscle activity, for AP initiation, AP propagation, and t-system excitation. Experimental observations of frequency-dependent length constant and membrane-phase properties in fast-twitch rat fibers could only be replicated by models that included t-system luminal resistances. Having quantified these resistances, the resulting models showed enhanced conduction velocity of passive current flow also implicating elevated AP propagation velocity. Furthermore, the resistances filter passive currents such that higher frequency current components would determine sarcolemma AP conduction velocity, whereas lower frequency components excite t-system APs. Because GM modulation affects only the low-frequency membrane impedance, the GM changes in active muscle would predominantly affect neuromuscular transmission and low-frequency t-system excitation while exerting little influence on the high-frequency process of sarcolemmal AP propagation. This physiological role of GM regulation was increased by high Cl− permeability, as in muscle endplate regions, and by increased extracellular [K+], as observed in working muscle. Thus, reduced GM at the onset of exercise would enhance t-system excitation and neuromuscular transmission, whereas elevated GM after sustained activity would inhibit these processes and thereby accentuate muscle fatigue. PMID:21670208

Down-regulation of angiotensin II receptor subtypes and desensitization of cyclic GMP production in neuroblastoma N1E-115 cells.

PubMed

Reagan, L P; Ye, X; Maretzski, C H; Fluharty, S J

1993-01-01

Murine neuroblastoma N1E-115 cells possess membranous receptors for the octapeptide angiotensin II (AngII) whose density is substantially increased by in vitro differentiation. Incubation of differentiated N1E-115 cells with AngII produced a rapid decrease in receptor density, but did not alter the affinity of these receptors for either 125I-AngII or the high-affinity antagonist 125I-[Sarc1,Ile8]-AngII. This apparent down-regulation was dose related with an ED50 of 1 nM, and maximal decreases of approximately 90% were obtained with 100 nM AngII. Receptor loss from differentiated cell membranes was unaffected by incubations of membranes obtained from agonist-exposed cells with non-hydrolyzable analogues of GTP for 60 min at 37 degrees C to ensure dissociation of the ligand. Partial loss of AngII receptors was apparent within 5 min of agonist exposure, whereas maximal declines were not observed until 30 min. This temporal pattern resulted from a preferential decrease in the AT1 receptor subtype during the first 5 min, followed by a decline in both AT1 and AT2 receptors with longer periods of agonist exposure. The loss of membranous receptors was reversible with partial recovery observed after 4 h, and with nearly full recovery observed 18 h after exposure of the cells to AngII. However, the long-term recovery of receptor density was blocked by the protein synthesis inhibitor, cycloheximide. The heptapeptide angiotensin III produced a similar down-regulation of receptors, and the high-affinity antagonist [Sarc1,Thr8]-AngII blocked agonist-induced down-regulation. Finally, the apparent loss of cell surface AngII receptors decreased the ability of AngII to stimulate cyclic GMP production within intact N1E-115 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi.

PubMed

Klayman, Lauren M; Wedegaertner, Philip B

2017-02-03

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

A Proteomic Approach to Characterize Protein Shedding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahram, Mamoun; Adkins, Joshua N.; Auberry, Deanna L.

2005-01-01

Shedding (i.e., proteolysis of ectodomains of membrane proteins) plays an important pathophysiological role. In order to study the feasibility of identifying shed proteins, we analyzed serum-free media of human mammary epithelial cells by mass spectrometry following induction of shedding by the phorbol ester, 4β-phorbol 12-myristate 13-acetate (PMA). Different means of sample preparation, including biotinylation of cell surface proteins, isolation of glycosylated proteins, and preparation of crude protein fraction, were carried out to develop the optimal method of sample processing. The collected proteins were digested with trypsin and analyzed by reversed-phase capillary liquid chromatography interfaced to an ion-trap mass spectrometer. Themore » resulting peptide spectra were interpreted using the program SEQUEST. Analyzing the sample containing the crude protein mixture without chemical modification or separation resulted in the greatest number of identifications, including putatively shed proteins. Overall, 93 membrane-associated proteins were identified including 57 that contain at least one transmembrane domain and 36 that indirectly associate with the extracellular surface of the plasma membrane. Of the 57 transmembrane proteins, 43 were identified by extracellular peptides providing strong evidence for them originating from regulated proteolysis or shedding processes. We combined results from the different experiments and used a peptide count method to estimate changes in protein abundance. Using this approach, we identified 2 proteins, syndecan-4 and hepatoma-derived growth factor, whose abundances increased in media of cells treated with PMA. We also detected proteins whose abundances decreased after PMA treatment such as 78-kDa glucose-regulated protein and calreticulin. Further analysis using immunoblotting validated the abundance changes for syndecan-4 and 78-kDa glucose-regulated protein as a result of PMA treatment. These results demonstrate that mass spectrometry can be used to identify low-abundance shed proteins and to estimate changes in protein abundances.« less

Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi*

PubMed Central

Klayman, Lauren M.; Wedegaertner, Philip B.

2017-01-01

Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM), where they are activated by cell surface G protein-coupled receptors. Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade, ultimately resulting in vesicle fission from the trans-Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid association mutant of the widely used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system. We show that GRK2ct-KERE cannot inhibit βγ function when expressed in cells, but recruitment to a specific membrane location recovers the ability of GRK2ct-KERE to inhibit βγ signaling. PM-recruited GRK2ct-KERE inhibits lysophosphatidic acid-induced phosphorylation of Akt, whereas Golgi-recruited GRK2ct-KERE inhibits cargo transport from the TGN to the PM. Moreover, we show that Golgi-recruited GRK2ct-KERE inhibits model basolaterally targeted but not apically targeted cargo delivery, for both PM-destined and secretory cargo, providing the first evidence of selectivity in terms of cargo transport regulated by βγ. Last, we show that Golgi fragmentation induced by ilimaquinone and nocodazole is blocked by βγ inhibition, demonstrating that βγ is a key regulator of multiple pathways that impact Golgi morphology. Thus, we have developed a new molecular tool, recruitable GRK2ct-KERE, to modulate βγ signaling at specific subcellular locations, and we demonstrate novel cargo selectivity for βγ regulation of TGN to PM transport and a novel role for βγ in mediating Golgi fragmentation. PMID:27994056

Disturbed vesicular trafficking of membrane proteins in prion disease.

PubMed

Uchiyama, Keiji; Miyata, Hironori; Sakaguchi, Suehiro

2013-01-01

The pathogenic mechanism of prion diseases remains unknown. We recently reported that prion infection disturbs post-Golgi trafficking of certain types of membrane proteins to the cell surface, resulting in reduced surface expression of membrane proteins and abrogating the signal from the proteins. The surface expression of the membrane proteins was reduced in the brains of mice inoculated with prions, well before abnormal symptoms became evident. Prions or pathogenic prion proteins were mainly detected in endosomal compartments, being particularly abundant in recycling endosomes. Some newly synthesized membrane proteins are delivered to the surface from the Golgi apparatus through recycling endosomes, and some endocytosed membrane proteins are delivered back to the surface through recycling endosomes. These results suggest that prions might cause neuronal dysfunctions and cell loss by disturbing post-Golgi trafficking of membrane proteins via accumulation in recycling endosomes. Interestingly, it was recently shown that delivery of a calcium channel protein to the cell surface was impaired and its function was abrogated in a mouse model of hereditary prion disease. Taken together, these results suggest that impaired delivery of membrane proteins to the cell surface is a common pathogenic event in acquired and hereditary prion diseases.

Electrodiffusion of lipids on membrane surfaces.

PubMed

Zhou, Y C

2012-05-28

Lateral translocation of lipids and proteins is a universal process on membrane surfaces. Local aggregation or organization of lipids and proteins can be induced when the random lateral motion is mediated by the electrostatic interactions and membrane curvature. Although the lateral diffusion rates of lipids on membranes of various compositions are measured and the electrostatic free energies of predetermined protein-membrane-lipid systems can be computed, the process of the aggregation and the evolution to the electrostatically favorable states remain largely undetermined. Here we propose an electrodiffusion model, based on the variational principle of the free energy functional, for the self-consistent lateral drift-diffusion of multiple species of charged lipids on membrane surfaces. Finite sizes of lipids are modeled to enforce the geometrical constraint of the lipid concentration on membrane surfaces. A surface finite element method is developed to appropriate the Laplace-Beltrami operators in the partial differential equations of the model. Our model properly describes the saturation of lipids on membrane surfaces, and correctly predicts that the MARCKS peptide can consistently sequester three multivalent phosphatidylinositol 4,5-bisphosphate lipids through its basic amino acid residues, regardless of a wide range of the percentage of monovalent phosphatidylserine in the membrane.

Electrodiffusion of lipids on membrane surfaces

NASA Astrophysics Data System (ADS)

Zhou, Y. C.

2012-05-01

Lateral translocation of lipids and proteins is a universal process on membrane surfaces. Local aggregation or organization of lipids and proteins can be induced when the random lateral motion is mediated by the electrostatic interactions and membrane curvature. Although the lateral diffusion rates of lipids on membranes of various compositions are measured and the electrostatic free energies of predetermined protein-membrane-lipid systems can be computed, the process of the aggregation and the evolution to the electrostatically favorable states remain largely undetermined. Here we propose an electrodiffusion model, based on the variational principle of the free energy functional, for the self-consistent lateral drift-diffusion of multiple species of charged lipids on membrane surfaces. Finite sizes of lipids are modeled to enforce the geometrical constraint of the lipid concentration on membrane surfaces. A surface finite element method is developed to appropriate the Laplace-Beltrami operators in the partial differential equations of the model. Our model properly describes the saturation of lipids on membrane surfaces, and correctly predicts that the MARCKS peptide can consistently sequester three multivalent phosphatidylinositol 4,5-bisphosphate lipids through its basic amino acid residues, regardless of a wide range of the percentage of monovalent phosphatidylserine in the membrane.

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

PubMed

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

2017-12-01

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

Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

PubMed Central

Defacque, Hélène; Bos, Evelyne; Garvalov, Boyan; Barret, Cécile; Roy, Christian; Mangeat, Paul; Shin, Hye-Won; Rybin, Vladimir; Griffiths, Gareth

2002-01-01

Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P2-binding proteins ezrin and/or moesin were essential for this process (Defacque et al., 2000b). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P2, and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P2-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane. PMID:11950931

Structural basis of sterol recognition and nonvesicular transport by lipid transfer proteins anchored at membrane contact sites.

PubMed

Tong, Junsen; Manik, Mohammad Kawsar; Im, Young Jun

2018-01-30

Membrane contact sites (MCSs) in eukaryotic cells are hotspots for lipid exchange, which is essential for many biological functions, including regulation of membrane properties and protein trafficking. Lipid transfer proteins anchored at membrane contact sites (LAMs) contain sterol-specific lipid transfer domains [StARkin domain (SD)] and multiple targeting modules to specific membrane organelles. Elucidating the structural mechanisms of targeting and ligand recognition by LAMs is important for understanding the interorganelle communication and exchange at MCSs. Here, we determined the crystal structures of the yeast Lam6 pleckstrin homology (PH)-like domain and the SDs of Lam2 and Lam4 in the apo form and in complex with ergosterol. The Lam6 PH-like domain displays a unique PH domain fold with a conserved N-terminal α-helix. The Lam6 PH-like domain lacks the basic surface for phosphoinositide binding, but contains hydrophobic patches on its surface, which are critical for targeting to endoplasmic reticulum (ER)-mitochondrial contacts. Structures of the LAM SDs display a helix-grip fold with a hydrophobic cavity and a flexible Ω1-loop as a lid. Ergosterol is bound to the pocket in a head-down orientation, with its hydrophobic acyl group located in the tunnel entrance. The Ω1-loop in an open conformation is essential for ergosterol binding by direct hydrophobic interaction. Structural comparison suggested that the sterol binding mode of the Lam2 SD2 is likely conserved among the sterol transfer proteins of the StARkin superfamily. Structural models of full-length Lam2 correlated with the sterol transport function at the membrane contact sites.

The interaction of polyglutamine peptides with lipid membranes is regulated by flanking sequences associated with huntingtin.

PubMed

Burke, Kathleen A; Kauffman, Karlina J; Umbaugh, C Samuel; Frey, Shelli L; Legleiter, Justin

2013-05-24

Huntington disease (HD) is caused by an expanded polyglutamine (poly(Q)) repeat near the N terminus of the huntingtin (htt) protein. Expanded poly(Q) facilitates formation of htt aggregates, eventually leading to deposition of cytoplasmic and intranuclear inclusion bodies containing htt. Flanking sequences directly adjacent to the poly(Q) domain, such as the first 17 amino acids on the N terminus (Nt17) and the polyproline (poly(P)) domain on the C-terminal side of the poly(Q) domain, heavily influence aggregation. Additionally, htt interacts with a variety of membraneous structures within the cell, and Nt17 is implicated in lipid binding. To investigate the interaction between htt exon1 and lipid membranes, a combination of in situ atomic force microscopy, Langmuir trough techniques, and vesicle permeability assays were used to directly monitor the interaction of a variety of synthetic poly(Q) peptides with different combinations of flanking sequences (KK-Q35-KK, KK-Q35-P10-KK, Nt17-Q35-KK, and Nt17-Q35-P10-KK) on model membranes and surfaces. Each peptide aggregated on mica, predominately forming extended, fibrillar aggregates. In contrast, poly(Q) peptides that lacked the Nt17 domain did not appreciably aggregate on or insert into lipid membranes. Nt17 facilitated the interaction of peptides with lipid surfaces, whereas the poly(P) region enhanced this interaction. The aggregation of Nt17-Q35-P10-KK on the lipid bilayer closely resembled that of a htt exon1 construct containing 35 repeat glutamines. Collectively, this data suggests that the Nt17 domain plays a critical role in htt binding and aggregation on lipid membranes, and this lipid/htt interaction can be further modulated by the presence of the poly(P) domain.

A Simple, Cost-Efficient Method to Separate Microalgal Lipids from Wet Biomass Using Surface Energy-Modified Membranes.

PubMed

Kwak, Moo Jin; Yoo, Youngmin; Lee, Han Sol; Kim, Jiyeon; Yang, Ji-Won; Han, Jong-In; Im, Sung Gap; Kwon, Jong-Hee

2016-01-13

For the efficient separation of lipid extracted from microalgae cells, a novel membrane was devised by introducing a functional polymer coating onto a membrane surface by means of an initiated chemical vapor deposition (iCVD) process. To this end, a steel-use-stainless (SUS) membrane was modified in a way that its surface energy was systemically modified. The surface modification by conformal coating of functional polymer film allowed for selective separation of oil-water mixture, by harnessing the tuned interfacial energy between each liquid phase and the membrane surface. The surface-modified membrane, when used with chloroform-based solvent, exhibited superb permeate flux, breakthrough pressure, and also separation yield: it allowed separation of 95.5 ± 1.2% of converted lipid (FAME) in the chloroform phase from the water/MeOH phase with microalgal debris. This result clearly supported that the membrane-based lipid separation is indeed facilitated by way of membrane being functionalized, enabling us to simplify the whole downstream process of microalgae-derived biodiesel production.

Composite fuel cell membranes

DOEpatents

Plowman, K.R.; Rehg, T.J.; Davis, L.W.; Carl, W.P.; Cisar, A.J.; Eastland, C.S.

1997-08-05

A bilayer or trilayer composite ion exchange membrane is described suitable for use in a fuel cell. The composite membrane has a high equivalent weight thick layer in order to provide sufficient strength and low equivalent weight surface layers for improved electrical performance in a fuel cell. In use, the composite membrane is provided with electrode surface layers. The composite membrane can be composed of a sulfonic fluoropolymer in both core and surface layers.

Composite fuel cell membranes

DOEpatents

Plowman, Keith R.; Rehg, Timothy J.; Davis, Larry W.; Carl, William P.; Cisar, Alan J.; Eastland, Charles S.

1997-01-01

A bilayer or trilayer composite ion exchange membrane suitable for use in a fuel cell. The composite membrane has a high equivalent weight thick layer in order to provide sufficient strength and low equivalent weight surface layers for improved electrical performance in a fuel cell. In use, the composite membrane is provided with electrode surface layers. The composite membrane can be composed of a sulfonic fluoropolymer in both core and surface layers.

Membrane Diffusion Occurs by Continuous-Time Random Walk Sustained by Vesicular Trafficking.

PubMed

Goiko, Maria; de Bruyn, John R; Heit, Bryan

2018-06-19

Diffusion in cellular membranes is regulated by processes that occur over a range of spatial and temporal scales. These processes include membrane fluidity, interprotein and interlipid interactions, interactions with membrane microdomains, interactions with the underlying cytoskeleton, and cellular processes that result in net membrane movement. The complex, non-Brownian diffusion that results from these processes has been difficult to characterize, and moreover, the impact of factors such as membrane recycling on membrane diffusion remains largely unexplored. We have used a careful statistical analysis of single-particle tracking data of the single-pass plasma membrane protein CD93 to show that the diffusion of this protein is well described by a continuous-time random walk in parallel with an aging process mediated by membrane corrals. The overall result is an evolution in the diffusion of CD93: proteins initially diffuse freely on the cell surface but over time become increasingly trapped within diffusion-limiting membrane corrals. Stable populations of freely diffusing and corralled CD93 are maintained by an endocytic/exocytic process in which corralled CD93 is selectively endocytosed, whereas freely diffusing CD93 is replenished by exocytosis of newly synthesized and recycled CD93. This trafficking not only maintained CD93 diffusivity but also maintained the heterogeneous distribution of CD93 in the plasma membrane. These results provide insight into the nature of the biological and biophysical processes that can lead to significantly non-Brownian diffusion of membrane proteins and demonstrate that ongoing membrane recycling is critical to maintaining steady-state diffusion and distribution of proteins in the plasma membrane. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Antimicrobial membrane surfaces via efficient polyethyleneimine immobilization and cationization

NASA Astrophysics Data System (ADS)

Qiu, Wen-Ze; Zhao, Zi-Shu; Du, Yong; Hu, Meng-Xin; Xu, Zhi-Kang

2017-12-01

Biofouling control is a major task in membrane separation processes for water treatment and biomedical applications. In this work, N-alkylated polyethylenimine (PEI) is facilely and efficiently introduced onto the membrane surfaces via the co-deposition of catechol (CCh) and PEI, followed by further grafting of PEIs (600 Da, 70 kDa and 750 kDa) and cationization with methyl iodide (CH3I). The physical and chemical properties of the constructed membrane surfaces are characterized with scanning electron microscopy, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, zeta potential and water contact angle measurements. Antibacterial assay reveals that the optimized membrane surfaces possess around 95% antibacterial efficiency against Gram-positive Staphylococcus aureus (S. aureus) with weak adhesion of bacteria cells after 24 h of bacterial contact. Additionally, the membrane surfaces also exhibit much enhanced antifouling property during the filtration of opposite charged bovine serum albumin (BSA). These results demonstrate a useful strategy for the surface modification of separation membranes by a kind of antimicrobial and antifouling coating.

A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

PubMed

Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

2011-05-01

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

Integrated self-organization of transitional ER and early Golgi compartments.

PubMed

Glick, Benjamin S

2014-02-01

COPII coated vesicles bud from an ER domain termed the transitional ER (tER), but the mechanism that clusters COPII vesicles at tER sites is unknown. tER sites are closely associated with early Golgi or pre-Golgi structures, suggesting that the clustering of nascent COPII vesicles could be achieved by tethering to adjacent membranes. This model challenges the prevailing view that COPII vesicles are clustered by a scaffolding protein at the ER surface. Although Sec16 was proposed to serve as such a scaffolding protein, recent data suggest that rather than organizing COPII into higher-order structures, Sec16 acts at the level of individual COPII vesicles to regulate COPII turnover. A plausible synthesis is that tER sites are created by tethering to Golgi membranes and are regulated by Sec16. Meanwhile, the COPII vesicles that bud from tER sites are thought to nucleate new Golgi cisternae. Thus, an integrated self-organization process may generate tER-Golgi units. © 2014 WILEY Periodicals, Inc.

The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stiles, Katie M.; Center for Oral Health Research, School of Dental Medicine University of Pennsylvania, Philadelphia, PA 19104; Milne, Richard S.B.

2008-03-30

During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated.more » Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.« less

Covalent attachment of phospholipid analogous polymers to modify a polymeric membrane surface: a novel approach.

PubMed

Xu, Zhi-Kang; Dai, Qing-Wen; Wu, Jian; Huang, Xiao-Jun; Yang, Qian

2004-02-17

A novel method for the surface modification of a microporous polypropylene membrane by tethering phospholipid analogous polymers (PAPs) is given, which includes the photoinduced graft polymerization of N,N-dimethylaminoethyl methacrylate (DMAEMA) and the ring-opening reaction of grafted poly-(DMAEMA) with 2-alkyloxy-2-oxo-1,3,2-dioxaphospholanes. Five 2-alkyloxy-2-oxo-1,3,2-dioxaphospholanes, containing octyloxy, dodecyloxy, tetradecyloxy, hexadecyloxy, and octadecyloxy groups in the molecular structure, were used to fabricate the PAP-modified polypropylene membranes. The attenuated total reflectance FT-IR spectra of the original, poly(DMAEMA)-grafted, and PAP-modified membranes confirmed the chemical changes on the membrane surface. Scanning electron microscope pictures showed that, compared with the original membrane, the surface porosities ofpoly(DMAEMA)-grafted and PAP-modified membranes were somewhat reduced. Water contact angles measured by the sessile drop method on PAP-modified membranes were slightly lower than that on the original polypropylene membrane, but higher than those on poly(DMAEMA)-grafted membranes with the exception of octyloxy-containing PAP-modified membranes. However, BSA adsorption experiments indicated that the five PAP-modified membranes had a much better protein-resistant property than the original polypropylene membrane and the poly(DMAEMA)-grafted membranes. For hexadecyloxy- and octadecyloxy-containing PAP-modified membranes, almost no protein adsorption was observed when the grafting degree was above 6 wt %. It was also found that the platelet adhesion was remarkably suppressed on the PAP-modified membranes. All these results demonstrate that the described approach is an effective way to improve the surface biocompatibility for polymeric membranes.

FAS grafted superhydrophobic ceramic membrane

NASA Astrophysics Data System (ADS)

Lu, Jun; Yu, Yun; Zhou, Jianer; Song, Lixin; Hu, Xingfang; Larbot, Andre

2009-08-01

The hydrophobic properties of γ-Al 2O 3 membrane have been obtained by grafting fluoroalkylsilane (FAS) on the surface of the membrane. The following grafting parameters were studied: the eroding time of the original membrane, the grafting time, the concentration of FAS solution and the multiplicity of grafting. Hydrophobicity of the membranes was characterized by contact angle (CA) measurement. The thermogravimetric analysis (TGA) was used to investigate the weight loss process (25-800 °C) of the fluoroalkylsilane grafted on Al 2O 3 powders under different grafting conditions. The morphologies of the membranes modified under different parameters were examined by field emission scanning electron microscopy (FE-SEM) and the surface roughness (Ra) was measured using white light interferometers. A needle-like structure was observed on the membrane surface after modification, which causes the change of Ra. On the results above, we speculated a model to describe the reaction between FAS and γ-Al 2O 3 membrane surface as well as the formed surface morphology.

Printing-assisted surface modifications of patterned ultrafiltration membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wardrip, Nathaniel C.; Dsouza, Melissa; Urgun-Demirtas, Meltem

Understanding and restricting microbial surface attachment will enhance wastewater treatment with membranes. We report a maskless lithographic patterning technique for the generation of patterned polymer coatings on ultrafiltration membranes. Polyethylene glycol, zwitterionic, or negatively charged hydrophilic polymer compositions in parallel- or perpendicular-striped patterns with respect to feed flow were evaluated using wastewater. Membrane fouling was dependent on the orientation and chemical composition of the coatings. Modifications reduced alpha diversity in the attached microbial community (Shannon indices decreased from 2.63 to 1.89) which nevertheless increased with filtration time. Sphingomonas species, which condition membrane surfaces and facilitate cellular adhesion, were depleted inmore » all modified membranes. Microbial community structure was significantly different between control, different patterns, and different chemistries. Lastly, this study broadens the tools for surface modification of membranes with polymer coatings and for understanding and optimization of antifouling surfaces.« less

Printing-assisted surface modifications of patterned ultrafiltration membranes

DOE PAGES

Wardrip, Nathaniel C.; Dsouza, Melissa; Urgun-Demirtas, Meltem; ...

2016-10-17

Understanding and restricting microbial surface attachment will enhance wastewater treatment with membranes. We report a maskless lithographic patterning technique for the generation of patterned polymer coatings on ultrafiltration membranes. Polyethylene glycol, zwitterionic, or negatively charged hydrophilic polymer compositions in parallel- or perpendicular-striped patterns with respect to feed flow were evaluated using wastewater. Membrane fouling was dependent on the orientation and chemical composition of the coatings. Modifications reduced alpha diversity in the attached microbial community (Shannon indices decreased from 2.63 to 1.89) which nevertheless increased with filtration time. Sphingomonas species, which condition membrane surfaces and facilitate cellular adhesion, were depleted inmore » all modified membranes. Microbial community structure was significantly different between control, different patterns, and different chemistries. Lastly, this study broadens the tools for surface modification of membranes with polymer coatings and for understanding and optimization of antifouling surfaces.« less

Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

PubMed

Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

2013-08-01

Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

Study of the Effect of Nanoparticles and Surface Morphology on Reverse Osmosis and Nanofiltration Membrane Productivity

PubMed Central

Fang, Yuming; Duranceau, Steven J.

2013-01-01

To evaluate the significance of reverse osmosis (RO) and nanofiltration (NF) surface morphology on membrane performance, productivity experiments were conducted using flat-sheet membranes and three different nanoparticles, which included SiO2, TiO2 and CeO2. In this study, the productivity rate was markedly influenced by membrane surface morphology. Atomic force microscopy (AFM) analysis of membrane surfaces revealed that the higher productivity decline rates associated with polyamide RO membranes as compared to that of a cellulose acetate NF membrane was due to the inherent ridge-and-valley morphology of the active layer. The unique polyamide active layer morphology was directly related to the surface roughness, and was found to contribute to particle accumulation in the valleys causing a higher flux decline than in smoother membranes. Extended RO productivity experiments using laboratory grade water and diluted pretreated seawater were conducted to compare the effect that different nanoparticles had on membrane active layers. Membrane flux decline was not affected by particle type when the feed water was laboratory grade water. On the other hand, membrane productivity was affected by particle type when pretreated diluted seawater served as feed water. It was found that CeO2 addition resulted in the least observable flux decline, followed by SiO2 and TiO2. A productivity simulation was conducted by fitting the monitored flux data into a cake growth rate model, where the model was modified using a finite difference method to incorporate surface thickness variation into the analysis. The ratio of cake growth term (k1) and particle back diffusion term (k2) was compared in between different RO and NF membranes. Results indicated that k2 was less significant for surfaces that exhibited a higher roughness. It was concluded that the valley areas of thin-film membrane surfaces have the ability to capture particles, limiting particle back diffusion. PMID:24956946

Optimization of fluorimetric lipid membrane biosensor sensitivity through manipulation of membrane structure and nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine concentration

NASA Astrophysics Data System (ADS)

Shrive, Jason D. A.; Krull, Ulrich J.

1995-01-01

In the work reported here, surface concentrations of 0.027 and 0.073 molecules nm-2 of the fluorescent membrane probe molecule nitrobenzoxadiazole dipalmitoylphosphatidylethanolamine (NBD-PE) were shown to yield optimum sensitivity for fluorimetric transduction of membrane structural perturbations for lipid membrane-based biosensor development. These optima were obtained through correlation of experimental data with theoretical predictions of optimum surface concentrations based on a model for NBD-PE self quenching previously published by our group. It was also determined that membrane structural heterogeneity improves the sensitivity of NBD-PE labeled membrane transducers. Together with fluorescence microscopy, observations of surface potential change upon compression or expansion of phosphatidylcholine (PC)/phosphatidic acid (PA) monolayers were used to qualitatively indicate the degree of structural heterogeneity in these membranes. It was determined that sub-microscopic domains must exist in microscopically homogeneous egg PC/egg PA membranes in order to facilitate the observed NBD-PE self-quenching responses upon alteration of bulk pH and therefore, membrane surface electrostatics and structure.

Atmospheric-pressure plasma activation and surface characterization on polyethylene membrane separator

NASA Astrophysics Data System (ADS)

Tseng, Yu-Chien; Li, Hsiao-Ling; Huang, Chun

2017-01-01

The surface hydrophilic activation of a polyethylene membrane separator was achieved using an atmospheric-pressure plasma jet. The surface of the atmospheric-pressure-plasma-treated membrane separator was found to be highly hydrophilic realized by adjusting the plasma power input. The variations in membrane separator chemical structure were confirmed by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Chemical analysis showed newly formed carbonyl-containing groups and high surface concentrations of oxygen-containing species on the atmospheric-pressure-plasma-treated polymeric separator surface. It also showed that surface hydrophilicity primarily increased from the polar component after atmospheric-pressure plasma treatment. The surface and pore structures of the polyethylene membrane separator were examined by scanning electron microscopy, revealing a slight alteration in the pore structure. As a result of the incorporation of polar functionalities by atmospheric-pressure plasma activation, the electrolyte uptake and electrochemical impedance of the atmospheric-pressure-plasma-treated membrane separator improved. The investigational results show that the separator surface can be controlled by atmospheric-pressure plasma surface treatment to tailor the hydrophilicity and enhance the electrochemical performance of lithium ion batteries.

Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

PubMed

Lim, HooiCheng; Yu, Chun-Ying; Jou, Tzuu-Shuh

2017-11-01

Establishment of apical-basal polarity, through correct targeting of polarity determinants to distinct domains of the plasma membrane, is a fundamental process for the development of functioning epithelial tubules. Here we report that galectin (Gal)-8 regulates apical-basal polarity of Madin-Darby canine kidney (MDCK) cells via apical targeting of 135-kDa glycoprotein (Gp135). Gal-8 interacts with newly synthesized Gp135 in a glycan-dependent manner. Gal-8 knockdown induces aberrant lumens at the lateral domain and mistargeting of Gp135 to this structure, thus disrupting the kidney epithelial polarity of MDCK cells, which organize lumens at the apical surface. The O -glycosylation deletion mutant of Gp135 phenocopies the effect of Gal-8 knockdown, which suggests that Gal-8 is the decoding machinery for the apical sorting signals of Gp135 residing at its O -glycosylation-rich region. Collectively, our results reveal a new role of Gal-8 in the development of luminal organs by regulating targeting of apical polarity protein Gp135.-Lim, H., Yu, C.-Y., Jou, T.-S. Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells. © FASEB.

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator

PubMed Central

Luciani, Alessandro; Villella, Valeria Rachela; Esposito, Speranza; Gavina, Manuela; Russo, Ilaria; Silano, Marco; Guido, Stefano; Pettoello-Mantovani, Massimo; Carnuccio, Rosa; Scholte, Bob; De Matteis, Antonella; Maiuri, Maria Chiara; Raia, Valeria; Luini, Alberto; Kroemer, Guido; Maiuri, Luigi

2012-01-01

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators. PMID:22874563

Targeting autophagy as a novel strategy for facilitating the therapeutic action of potentiators on ΔF508 cystic fibrosis transmembrane conductance regulator.

PubMed

Luciani, Alessandro; Villella, Valeria Rachela; Esposito, Speranza; Gavina, Manuela; Russo, Ilaria; Silano, Marco; Guido, Stefano; Pettoello-Mantovani, Massimo; Carnuccio, Rosa; Scholte, Bob; De Matteis, Antonella; Maiuri, Maria Chiara; Raia, Valeria; Luini, Alberto; Kroemer, Guido; Maiuri, Luigi

2012-11-01

Channel activators (potentiators) of cystic fibrosis (CF) transmembrane conductance regulator (CFTR), can be used for the treatment of the small subset of CF patients that carry plasma membrane-resident CFTR mutants. However, approximately 90% of CF patients carry the misfolded ΔF508-CFTR and are poorly responsive to potentiators, because ΔF508-CFTR is intrinsically unstable at the plasma membrane (PM) even if rescued by pharmacological correctors. We have demonstrated that human and mouse CF airways are autophagy deficient due to functional sequestration of BECN1 and that the tissue transglutaminase-2 inhibitor, cystamine, or antioxidants restore BECN1-dependent autophagy and reduce SQSTM1/p62 levels, thus favoring ΔF508-CFTR trafficking to the epithelial surface. Here, we investigated whether these treatments could facilitate the beneficial action of potentiators on ΔF508-CFTR homozygous airways. Cystamine or the superoxide dismutase (SOD)/catalase-mimetic EUK-134 stabilized ΔF508-CFTR at the plasma membrane of airway epithelial cells and sustained the expression of CFTR at the epithelial surface well beyond drug withdrawal, overexpressing BECN1 and depleting SQSTM1. This facilitates the beneficial action of potentiators in controlling inflammation in ex vivo ΔF508-CFTR homozygous human nasal biopsies and in vivo in mouse ΔF508-CFTR lungs. Direct depletion of Sqstm1 by shRNAs in vivo in ΔF508-CFTR mice synergized with potentiators in sustaining surface CFTR expression and suppressing inflammation. Cystamine pre-treatment restored ΔF508-CFTR response to the CFTR potentiators genistein, Vrx-532 or Vrx-770 in freshly isolated brushed nasal epithelial cells from ΔF508-CFTR homozygous patients. These findings delineate a novel therapeutic strategy for the treatment of CF patients with the ΔF508-CFTR mutation in which patients are first treated with cystamine and subsequently pulsed with CFTR potentiators.

Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells.

PubMed Central

Pörn, M I; Slotte, J P

1990-01-01

Plasma-membrane sphingomyelin appears to be one of the major determinants of the preferential allocation of cell cholesterol into the plasma-membrane compartment, since removal of sphingomyelin leads to a dramatic redistribution of cholesterol within the cell [Slotte & Bierman (1988) Biochem. J. 250, 653-658]. In the present study we examined the long-term effects of sphingomyelin degradation on cholesterol redistribution in cells and determined the reversibility of the process. In a human lung fibroblast-cell line, removal of 80% of the sphingomyelin led to a rapid and transient up-regulation (3-fold) of acyl-CoA:cholesterol acyltransferase (ACAT) activity, and also, within 30 h, to the translocation of about 50% of the cell non-esterified cholesterol from a cholesterol oxidase-susceptible compartment (i.e. the cell surface) to oxidase-resistant compartments. At 49 h after the initial sphingomyelin degradation, the cell sphingomyelin level was back to 45% of the control level, and the direction of cell cholesterol flow was toward the cell surface, although the original distribution was not achieved. In a transformed neuroblastoma cell line (SH-SY5Y), the depletion of sphingomyelin led to a similarly rapid and transient up-regulation of ACAT activity, and to the translocation of about 25% of cell-surface cholesterol into internal membranes (within 3 h). The flow of cholesterol back to the cholesterol oxidase-susceptible pool was rapid, and a pretreatment cholesterol distribution was reached within 20-49 h. Also, the resynthesis of sphingomyelin was faster in SH-SY5Y neuroblastoma cells and reached control levels within 24 h. The findings of the present study show that the cellular redistribution of cholesterol, as induced by sphingomyelin degradation, is reversible and suggest that the normalization of cellular cholesterol distribution is linked to the re-synthesis of sphingomyelin. PMID:2222406

Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis.

PubMed

Dzierlenga, Anika L; Cherrington, Nathan J

2018-03-01

Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance-associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization. © 2018 Wiley Periodicals, Inc.

Method and apparatus for determining the hydraulic conductivity of earthen material

DOEpatents

Sisson, James B.; Honeycutt, Thomas K.; Hubbell, Joel M.

1996-01-01

An earthen material hydraulic conductivity determining apparatus includes, a) a semipermeable membrane having a fore earthen material bearing surface and an opposing rear liquid receiving surface; b) a pump in fluid communication with the semipermeable membrane rear surface, the pump being capable of delivering liquid to the membrane rear surface at a plurality of selected variable flow rates or at a plurality of selected variable pressures; c) a liquid reservoir in fluid communication with the pump, the liquid reservoir retaining a liquid for pumping to the membrane rear surface; and d) a pressure sensor in fluid communication with the membrane rear surface to measure pressure of liquid delivered to the membrane by the pump. Preferably, the pump comprises a pair of longitudinally opposed and aligned syringes which are operable to simultaneously fill one syringe while emptying the other. Methods of determining the hydraulic conductivity of earthen material are also disclosed.

Method and apparatus for determining the hydraulic conductivity of earthen material

DOEpatents

Sisson, J.B.; Honeycutt, T.K.; Hubbell, J.M.

1996-05-28

An earthen material hydraulic conductivity determining apparatus includes: (a) a semipermeable membrane having a fore earthen material bearing surface and an opposing rear liquid receiving surface; (b) a pump in fluid communication with the semipermeable membrane rear surface, the pump being capable of delivering liquid to the membrane rear surface at a plurality of selected variable flow rates or at a plurality of selected variable pressures; (c) a liquid reservoir in fluid communication with the pump, the liquid reservoir retaining a liquid for pumping to the membrane rear surface; and (d) a pressure sensor in fluid communication with the membrane rear surface to measure pressure of liquid delivered to the membrane by the pump. Preferably, the pump comprises a pair of longitudinally opposed and aligned syringes which are operable to simultaneously fill one syringe while emptying the other. Methods of determining the hydraulic conductivity of earthen material are also disclosed. 15 figs.

C2 Domain of Protein Kinase Cα: Elucidation of the Membrane Docking Surface by Site-Directed Fluorescence and Spin Labeling†

PubMed Central

Kohout, Susy C.; Corbalán-García, Senena; Gómez-Fernández, Juan C.; Falke, Joseph J.

2013-01-01

The C2 domain is a conserved signaling motif that triggers membrane docking in a Ca2+-dependent manner, but the membrane docking surfaces of many C2 domains have not yet been identified. Two extreme models can be proposed for the docking of the protein kinase Cα (PKCα) C2 domain to membranes. In the parallel model, the membrane-docking surface includes the Ca2+ binding loops and an anion binding site on β-strands 3–4, such that the β-strands are oriented parallel to the membrane. In the perpendicular model, the docking surface is localized to the Ca2+ binding loops and the β-strands are oriented perpendicular to the membrane surface. The present study utilizes site-directed fluorescence and spin-labeling to map out the membrane docking surface of the PKCα C2 domain. Single cysteine residues were engineered into 18 locations scattered over all regions of the protein surface, and were used as attachment sites for spectroscopic probes. The environmentally sensitive fluorescein probe identified positions where Ca2+ activation or membrane docking trigger measurable fluorescence changes. Ca2+ binding was found to initiate a global conformational change, while membrane docking triggered the largest fluorescein environmental changes at labeling positions on the three Ca2+ binding loops (CBL), thereby localizing these loops to the membrane docking surface. Complementary EPR power saturation measurements were carried out using a nitroxide spin probe to determine a membrane depth parameter, Φ, for each spin-labeled mutant. Positive membrane depth parameters indicative of membrane insertion were found for three positions, all located on the Ca2+ binding loops: N189 on CBL 1, and both R249 and R252 on CBL 3. In addition, EPR power saturation revealed that five positions near the anion binding site are partially protected from collisions with an aqueous paramagnetic probe, indicating that the anion binding site lies at or near the surface of the headgroup layer. Together, the fluorescence and EPR results indicate that the Ca2+ first and third Ca2+ binding loops insert directly into the lipid headgroup region of the membrane, and that the anion binding site on β-strands 3–4 lies near the headgroups. The data support a model in which the β-strands are tilted toward the parallel orientation relative to the membrane surface. PMID:12564928

Surface expression of NMDA receptor changes during memory consolidation in the crab Neohelice granulata

PubMed Central

Hepp, Yanil; Salles, Angeles; Carbo-Tano, Martin

2016-01-01

The aim of the present study was to analyze the surface expression of the NMDA-like receptors during the consolidation of contextual learning in the crab Neohelice granulata. Memory storage is based on alterations in the strength of synaptic connections between neurons. The glutamatergic synapses undergo various forms of N-methyl-D aspartate receptor (NMDAR)-dependent changes in strength, a process that affects the abundance of other receptors at the synapse and underlies some forms of learning and memory. Here we propose a direct regulation of the NMDAR. Changes in NMDAR's functionality might be induced by the modification of the subunit's expression or cellular trafficking. This trafficking does not only include NMDAR's movement between synaptic and extra-synaptic localizations but also the cycling between intracellular compartments and the plasma membrane, a process called surface expression. Consolidation of contextual learning affects the surface expression of the receptor without affecting its general expression. The surface expression of the GluN1 subunit of the NMDAR is down-regulated immediately after training, up-regulated 3 h after training and returns to naïve and control levels 24 h after training. The changes in NMDAR surface expression observed in the central brain are not seen in the thoracic ganglion. A similar increment in surface expression of GluN1 in the central brain is observed 3 h after administration of the competitive GABAA receptor antagonist, bicuculline. These consolidation changes are part of a plasticity event that first, during the down-regulation, stabilizes the trace and later, at 3-h post-training, changes the threshold for synapse activation. PMID:27421895

Structural Fine-Tuning of MIT-Interacting Motif 2 (MIM2) and Allosteric Regulation of ESCRT-III by Vps4 in Yeast.

PubMed

Kojima, Rieko; Obita, Takayuki; Onoue, Kousuke; Mizuguchi, Mineyuki

2016-06-05

The endosomal sorting complex required for transport (ESCRT) facilitates roles in membrane remodeling, such as multivesicular body biogenesis, enveloped virus budding and cell division. In yeast, Vps4 plays a crucial role in intraluminal vesicle formation by disassembling ESCRT proteins. Vps4 is recruited by ESCRT-III proteins to the endosomal membrane through the interaction between the microtubule interacting and trafficking (MIT) domain of Vps4 and the C-terminal MIT-interacting motif (MIM) of ESCRT-III proteins. Here, we have determined the crystal structure of Vps4-MIT in a complex with Vps20, a member of ESCRT-III, and revealed that Vps20 adopts a unique MIM2 conformation. Based on structural comparisons with other known MIM2s, we have refined the consensus sequence of MIM2. We have shown that another ESCRT-III protein, Ist1, binds to Vps4-MIT via its C-terminal MIM1 with higher affinity than Vps2, but lacks MIM2 by surface plasmon resonance. Surprisingly, the Ist1 MIM1 competed with the MIM2 of Vfa1, a regulator of Vps4, for binding to Vps4-MIT, even though these MIMs bind in non-overlapping sites on the MIT. These findings provide insight into the allosteric recognition of MIMs of ESCRT-III by Vps4 and also the regulation of ESCRT machinery at the last step of membrane remodeling. Copyright © 2016 Elsevier Ltd. All rights reserved.

Omniphobic Membrane for Robust Membrane Distillation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, SH; Nejati, S; Boo, C

2014-11-01

In this work, we fabricate an omniphobic microporous membrane for membrane distillation (MD) by modifying a hydrophilic glass fiber membrane with silica nanoparticles followed by surface fluorination and polymer coating. The modified glass fiber membrane exhibits an anti-wetting property not only against water but also against low surface tension organic solvents that easily wet a hydrophobic polytetrafluoroethylene (PTFE) membrane that is commonly used in MD applications. By comparing the performance of the PTFE and omniphobic membranes in direct contact MD experiments in the presence of a surfactant (sodium dodecyl sulfate, SDS), we show that SDS wets the hydrophobic PTFE membranemore » but not the omniphobic membrane. Our results suggest that omniphobic membranes are critical for MD applications with feed waters containing surface active species, such as oil and gas produced water, to prevent membrane pore wetting.« less

NAc Shell Arc/Arg3.1 Protein Mediates Reconsolidation of Morphine CPP by Increased GluR1 Cell Surface Expression: Activation of ERK-Coupled CREB is Required

PubMed Central

Lv, Xiu-Fang; Sun, Lin-Lin; Han, Ji-Sheng

2015-01-01

Background: Relapse into drug abuse evoked by reexposure to the drug-associated context has been a primary problem in the treatment of drug addiction. Disrupting the reconsolidation of drug-related context memory would therefore limit the relapse susceptibility. Methods: Morphine conditioned place preference (CPP) was used to assess activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and correlative molecule expression in the Nucleus accumbens (NAc) shell during the reconsolidation of morphine CPP. U0126 and Arc/Arg3.1 antisense oligodeoxynucleotide were adapted to evaluate the role and the underlying mechanism of Arc/Arg3.1 during the reconsolidation. Results: The retrieval of morphine CPP in rats specifically increased the Arc/Arg3.1 protein level in the NAc shell, accompanied simultaneously by increases in the phosphorylation of extracellular signal-regulated kinase1/2 (pERK1/2), the phosphorylation of Cyclic Adenosine monophosphate (cAMP) response element-binding (pCREB), and the up-regulation of the membrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors GluR1 subunit level. Intra-NAc shell infusion U0126, an inhibitor of the Mitogen-activated protein kinase kinase (MEK), prevented the retrieval-induced up-regulation of pERK1/2, pCREB, Arc/Arg3.1, and membrane GluR1 immediately after retrieval of morphine CPP. The effect of disrupting the reconsolidation of morphine CPP by U0126 could last for at least 14 days, and could not be evoked by a priming injection of morphine. Furthermore, the specific knockdown of Arc/Arg3.1 in the NAc shell decreased the membrane GluR1 level, and impaired both the reconsolidation and the reinstatement of morphine CPP. Conclusions: Arc/Arg3.1 in the NAc shell mediates the reconsolidation of morphine-associated context memory via up-regulating the level of membrane of GluR1, for which the local activation of the ERK-CREB signal pathway, as an upstream mechanism of Arc/Arg3.1, is required. PMID:25746394

[Effect of Membrane Wettability on Membrane Fouling and Chemical Durability of SPG Membranes].

PubMed

Zhang, Jing; Xiao, Tai-min; Zhang, Jing; Cao, Li-ya; Du, Ya-wei; Liu, Chun; Zhang, Lei

2015-05-01

Shirasu porous glass (SPG) membranes have been applied for microbubble aeration in aerobic wastewater treatment. In the present study, both hydrophilic and hydrophobic SPG membranes were used in a microbubble-aerated biofilm reactor with online chemical cleaning, and their membrane fouling and chemical durability were determined to be strongly dependent on the membrane wettability. The fouling layer formed on the surface of both membranes was confirmed to be mainly organic fouling, and the hydrophobic membrane showed a relatively stronger resistance to the organic fouling. The severe chemical corrosion of the hydrophilic membrane was observed due to exposure to the alkaline sodium hypochlorite solution used for chemical cleaning, which resulted in significant increases in the median pore diameter and the porosity. On the other hand, the pore structure of the hydrophobic membrane changed slightly when exposed to the alkaline sodium hypochlorite solution, suggesting its strong alkali-resistance due to the non-wetting surface. However, the surface hydrophobic groups of hydrophobic membrane could be oxidized by sodium hypochlorite solution, resulting in more wettable membrane surface. The hydrophobic membrane also showed better performance in the respects of oxygen transfer, contaminant removal and energy-saving. Therefore, the hydrophobic membrane seemed more appropriate to be applied for microbubble aeration in aerobic wastewater treatment process.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

PubMed

Valkova, Christina; Albrizio, Marina; Röder, Ira V; Schwake, Michael; Betto, Romeo; Rudolf, Rüdiger; Kaether, Christoph

2011-01-11

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Engineering the Re-Entrant Hierarchy and Surface Energy of PDMS-PVDF Membrane for Membrane Distillation Using a Facile and Benign Microsphere Coating.

PubMed

Lee, Eui-Jong; Deka, Bhaskar Jyoti; Guo, Jiaxin; Woo, Yun Chul; Shon, Ho Kyong; An, Alicia Kyoungjin

2017-09-05

To consolidate the position of membrane distillation (MD) as an emerging membrane technology that meets global water challenges, it is crucial to develop membranes with ideal material properties. This study reports a facile approach for a polyvinylidene fluoride (PVDF) membrane surface modification that is achieved through the coating of the surface with poly(dimethylsiloxane) (PDMS) polymeric microspheres to lower the membrane surface energy. The hierarchical surface of the microspheres was built without any assistance of a nano/microcomposite by combining the rapid evaporation of tetrahydrofuran (THF) and the phase separation from condensed water vapor. The fabricated membrane exhibited superhydrophobicity-a high contact angle of 156.9° and a low contact-angle hysteresis of 11.3°-and a high wetting resistance to seawater containing sodium dodecyl sulfate (SDS). Compared with the control PVDF-hexafluoropropylene (HFP) single-layer nanofiber membrane, the proposed fabricated membrane with the polymeric microsphere layer showed a smaller pore size and higher liquid entry pressure (LEP). When it was tested for the direct-contact MD (DCMD) in terms of the desalination of seawater (3.5% of NaCl) containing SDS of a progressively increased concentration, the fabricated membrane showed stable desalination and partial wetting for the 0.1 and 0.2 mM SDS, respectively.

Antifouling enhancement of polysulfone/TiO2 nanocomposite separation membrane by plasma etching

NASA Astrophysics Data System (ADS)

Chen, Z.; Yin, C.; Wang, S.; Ito, K.; Fu, Q. M.; Deng, Q. R.; Fu, P.; Lin, Z. D.; Zhang, Y.

2017-01-01

A polysulfone/TiO2 nanocomposite membrane was prepared via casting method, followed by the plasma etching of the membrane surface. Doppler broadened energy spectra vs. positron incident energy were employed to elucidate depth profiles of the nanostructure for the as-prepared and treated membranes. The results confirmed that the near-surface of the membrane was modified by the plasma treatment. The antifouling characteristics for the membranes, evaluated using the degradation of Rhodamin B, indicated that the plasma treatment enhances the photo catalytic ability of the membrane, suggesting that more TiO2 nanoparticles are exposed at the membrane surface after the plasma treatment as supported by the positron result.

[Observation on eggs of Oncomelania hupensis hupensis with scanning electron microscope].

PubMed

Xia, Q B; Yuan, Y B; Liu, B; Tan, P P

2001-01-01

To observe the structure of the mud hull packed Oncomelania eggs and the surface structure of colloid membrane called the third grade membrane of eggs. Scanning electron microscopy was used to observe Oncomelania snail eggs with integral mud hull collected from eastern Dongting Lake. The mud hull of eggs was made of unshapen small humification combined with earth granules with a diameter of 2.6-9.2 microns. The mud hull in 60 um thickness was honeycomb-like in shape with many small holes and small folds on the wall. There were many round or irregularly round hollownesses on the inner layer of mud hull that contacts colloid membrane but no hole through mud hull. There were some protein fiber networks covering on the colloid membrane and apophysis. The structure of the mud hull showed that the exchange of matter was maintained between eggs and outside, and the mud hull is of great importance to regulating temperature and moisture for the growth of eggs by preventing hydrosoluble substances from penetrating into eggs. The protein fiber networks act on gluing mud hull and buffering outside power. The dense glue membrane might be a main barricade to prevent pharmaceutical molecules from penetrating into eggs.

VANGL2 regulates membrane trafficking of MMP14 to control cell polarity and migration.

PubMed

Williams, B Blairanne; Cantrell, V Ashley; Mundell, Nathan A; Bennett, Andrea C; Quick, Rachel E; Jessen, Jason R

2012-05-01

Planar cell polarity (PCP) describes the polarized orientation of cells within the plane of a tissue. Unlike epithelial PCP, the mechanisms underlying PCP signaling in migrating cells remain undefined. Here, the establishment of PCP must be coordinated with dynamic changes in cell adhesion and extracellular matrix (ECM) organization. During gastrulation, the membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) is required for PCP and convergence and extension cell movements. We report that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell-surface availability of MMP14 in manner that is dependent on focal adhesion kinase. We demonstrate that zebrafish trilobite/vangl2 mutant embryos exhibit increased Mmp14 activity and decreased ECM. Furthermore, in vivo knockdown of Mmp14 partially rescues the Vangl2 loss-of-function convergence and extension phenotype. This study identifies a mechanism linking VANGL2 with MMP14 trafficking and suggests that establishment of PCP in migrating gastrula cells requires regulated proteolytic degradation or remodeling of the ECM. Our findings implicate matrix metalloproteinases as downstream effectors of PCP and suggest a broadly applicable mechanism whereby VANGL2 affects diverse morphogenetic processes.

Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: implications for regulation of BMP signalling.

PubMed

Lopez-Escobar, Beatriz; De Felipe, Beatriz; Sanchez-Alcazar, Jose Antonio; Sasaki, Takako; Copp, Andrew J; Ybot-Gonzalez, Patricia

2012-11-01

The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β, and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. Copyright © 2012 Wiley Periodicals, Inc.

Laminin and integrin expression in the ventral ectodermal ridge of the mouse embryo: implications for regulation of BMP signalling

PubMed Central

Lopez-Escobar, Beatriz; de Felipe, Beatriz; Sanchez-Alcazar, Jose Antonio; Sasaki, Takako; Copp, Andrew J.; Ybot-Gonzalez, Patricia

2013-01-01

Background The ventral ectodermal ridge (VER) is an important signalling centre in the mouse tail-bud following completion of gastrulation. BMP regulation is essential for VER function, but how these signals are transmitted between adjacent tissues is unclear. Results We investigated the idea that extracellular matrix components might be involved, using immunohistochemistry and in situ hybridisation to detect all known α, β and γ laminin chains and their mRNAs in the early tail bud. We identified an apparently novel laminin variant, comprising α5, β3 and γ2 chains, as a major component of the VER basement membrane at E9.5. Strikingly, only the mRNAs for these chains were co-expressed in VER cells, suggesting that lamin532 may be the sole basement membrane laminin at this stage. Since α6 integrin was also expressed in VER cells, this raises the possibility of cell-matrix interactions regulating BMP signalling at this site of caudal morphogenesis. Conclusions Laminin532 could interact with α6-containing integrin to direct differentiation of the specialised VER cells from surface ectoderm. PMID:22911573

Surface glycosylation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) membrane for selective adsorption of low-density lipoprotein.

PubMed

Wang, Wei; Lan, Ping

2014-01-01

A novel method of constructing a glycosylated surface on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] membrane surface for the selective adsorption of low-density lipoprotein (LDL) was developed, which involved the photoinduced graft polymerization of acrylic acid followed by the chemical binding of carboxyl groups with glucosamine in the presence of 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride and N-hydroxy-succinimide. The chemical structures of the fabricated membranes were characterized by attenuated total reflectance Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy. Zeta potential and water contact angle measurements were performed to investigate the surface charge and wettability of the membranes, respectively. An enzyme linked immunosorbent assay was used to measure the LDL adsorption on the plain and modified membrane surfaces. It was found that the surface glycosylation of P(3HB-co-4HB) membrane greatly enhanced the affinity interactions with LDL and the absorbed LDL could be easily desorbed with eluents, indicating a specific and reversible binding of LDL to the surface. Furthermore, the hemocompatibility of glycosylated membrane was improved as examined by platelet adhesion. The results suggest that the glycosylated P(3HB-co-4HB) membrane is promising for application in LDL apheresis therapy.

The plasma membrane as a capacitor for energy and metabolism

PubMed Central

Ray, Supriyo; Kassan, Adam; Busija, Anna R.; Rangamani, Padmini

2016-01-01

When considering which components of the cell are the most critical to function and physiology, we naturally focus on the nucleus, the mitochondria that regulate energy and apoptotic signaling, or other organelles such as the endoplasmic reticulum, Golgi, ribosomes, etc. Few people will suggest that the membrane is the most critical element of a cell in terms of function and physiology. Those that consider the membrane critical will point to its obvious barrier function regulated by the lipid bilayer and numerous ion channels that regulate homeostatic gradients. What becomes evident upon closer inspection is that not all membranes are created equal and that there are lipid-rich microdomains that serve as platforms of signaling and a means of communication with the intracellular environment. In this review, we explore the evolution of membranes, focus on lipid-rich microdomains, and advance the novel concept that membranes serve as “capacitors for energy and metabolism.” Within this framework, the membrane then is the primary and critical regulator of stress and disease adaptation of the cell. PMID:26771520

Effect of incorporating graphene oxide and surface imprinting on polysulfone membranes on flux, hydrophilicity and rejection of salt and polycyclic aromatic hydrocarbons from water

NASA Astrophysics Data System (ADS)

Kibechu, Rose Waithiegeni; Ndinteh, Derek Tantoh; Msagati, Titus Alfred Makudali; Mamba, Bhekie Briliance; Sampath, S.

2017-08-01

We report a significant enhancement of hydrophillity of polysulfone (Psf) membranes after modification with graphene oxide (GO) as a filler followed by surface imprinting on the surface of GO/Psf composite imprinted membranes (CIMs). The surface imprinting on the GO-Psf membrane was employed in order to enhance its selectivity towards polycyclic aromatic hydrocarbons (PAHs) in water. The CIMs were prepared through a process of phase inversion of a mixture of graphene oxide and polysulfone (Psf) in N-methylpyrrolidone (NMP). Fourier-transform spectroscopy (FT-IR) of the imprinted showed new peaks at 935 cm-1 and 1638 cm-1 indicating success in surface imprinting on the GO-Psf membrane. The CIM also showed improvement in flux from 8.56 LM-2 h-1 of unmodified polysulfone membrane to 15.3 LM-2 h-1 in the CIM, salt rejection increased from 57.2 ± 4.2% of polysulfone membrane to 76 ± 4.5%. The results obtained from the contact angle measurements showed a decrease with increase in GO content from 72 ± 2.7% of neat polysulfone membrane to 62.3 ± 2.1% of CIM indicating an improvement in surface hydrophilicity. The results from this study shows that, it is possible to improve the hydrophilicity of the membranes without affecting the performance of the membranes.

PES Surface Modification Using Green Chemistry: New Generation of Antifouling Membranes.

PubMed

Nady, Norhan

2016-04-18

A major limitation in using membrane-based separation processes is the loss of performance due to membrane fouling. This drawback can be addressed thanks to surface modification treatments. A new and promising surface modification using green chemistry has been recently investigated. This modification is carried out at room temperature and in aqueous medium using green catalyst (enzyme) and nontoxic modifier, which can be safely labelled "green surface modification". This modification can be considered as a nucleus of new generation of antifouling membranes and surfaces. In the current research, ferulic acid modifier and laccase bio-catalyst were used to make poly(ethersulfone) (PES) membrane less vulnerable to protein adsorption. The blank and modified PES membranes are evaluated based on e.g., their flux and protein repellence. Both the blank and the modified PES membranes (or laminated PES on silicon dioxide surface) are characterized using many techniques e.g., SEM, EDX, XPS and SPM, etc. The pure water flux of the most modified membranes was reduced by 10% on average relative to the blank membrane, and around a 94% reduction in protein adsorption was determined. In the conclusions section, a comparison between three modifiers-ferulic acid, and two other previously used modifiers (4-hydroxybenzoic acid and gallic acid)-is presented.

PES Surface Modification Using Green Chemistry: New Generation of Antifouling Membranes

PubMed Central

Nady, Norhan

2016-01-01

A major limitation in using membrane-based separation processes is the loss of performance due to membrane fouling. This drawback can be addressed thanks to surface modification treatments. A new and promising surface modification using green chemistry has been recently investigated. This modification is carried out at room temperature and in aqueous medium using green catalyst (enzyme) and nontoxic modifier, which can be safely labelled “green surface modification”. This modification can be considered as a nucleus of new generation of antifouling membranes and surfaces. In the current research, ferulic acid modifier and laccase bio-catalyst were used to make poly(ethersulfone) (PES) membrane less vulnerable to protein adsorption. The blank and modified PES membranes are evaluated based on e.g., their flux and protein repellence. Both the blank and the modified PES membranes (or laminated PES on silicon dioxide surface) are characterized using many techniques e.g., SEM, EDX, XPS and SPM, etc. The pure water flux of the most modified membranes was reduced by 10% on average relative to the blank membrane, and around a 94% reduction in protein adsorption was determined. In the conclusions section, a comparison between three modifiers—ferulic acid, and two other previously used modifiers (4-hydroxybenzoic acid and gallic acid)—is presented. PMID:27096873

Transcellular tunnel dynamics: Control of cellular dewetting by actomyosin contractility and I-BAR proteins.

PubMed

Lemichez, Emmanuel; Gonzalez-Rodriguez, David; Bassereau, Patricia; Brochard-Wyart, Françoise

2013-03-01

Dewetting is the spontaneous withdrawal of a liquid film from a non-wettable surface by nucleation and growth of dry patches. Two recent reports now propose that the principles of dewetting explain the physical phenomena underpinning the opening of transendothelial cell macroaperture (TEM) tunnels, referred to as cellular dewetting. This was discovered by studying a group of bacterial toxins endowed with the property of corrupting actomyosin cytoskeleton contractility. For both liquid and cellular dewetting, the growth of holes is governed by a competition between surface forces and line tension. We also discuss how the dynamics of TEM opening and closure represent remarkable systems to investigate actin cytoskeleton regulation by sensors of plasma membrane curvature and investigate the impact on membrane tension and the role of TEM in vascular dysfunctions. Copyright © 2013 Soçiété Française des Microscopies and Soçiété de Biologie Cellulaire de France.

The Effect of Size and Species on Lens Intracellular Hydrostatic Pressure

PubMed Central

Gao, Junyuan; Sun, Xiurong; Moore, Leon C.; Brink, Peter R.; White, Thomas W.; Mathias, Richard T.

2013-01-01

Purpose. Previous experiments showed that mouse lenses have an intracellular hydrostatic pressure that varied from 335 mm Hg in central fibers to 0 mm Hg in surface cells. Model calculations predicted that in larger lenses, all else equal, pressure should increase as the lens radius squared. To test this prediction, lenses of different radii from different species were studied. Methods. All studies were done in intact lenses. Intracellular hydrostatic pressures were measured with a microelectrode-manometer–based system. Membrane conductances were measured by frequency domain impedance analysis. Intracellular Na+ concentrations were measured by injecting the Na+-sensitive dye sodium-binding benzofuran isophthalate. Results. Intracellular hydrostatic pressures were measured in lenses from mice, rats, rabbits, and dogs with radii (cm) 0.11, 0.22, 0.49, and 0.57, respectively. In each species, pressure varied from 335 ± 6 mm Hg in central fiber cells to 0 mm Hg in surface cells. Further characterization of transport in lenses from mice and rats showed that the density of fiber cell gap junction channels was approximately the same, intracellular Na+ concentrations varied from 17 mM in central fiber cells to 7 mM in surface cells, and intracellular voltages varied from −45 mV in central fiber cells to −60 mV in surface cells. Fiber cell membrane conductance was a factor of 2.7 times larger in mouse than in rat lenses. Conclusions. Intracellular hydrostatic pressure is an important physiological parameter that is regulated in lenses from these different species. The most likely mechanism of regulation is to reduce the density of open Na+-leak channels in fiber cells of larger lenses. PMID:23211824

Conductive super-hydrophobic surfaces of polyaniline modified porous anodic alumina membranes.

PubMed

Chen, Xinhua; Chen, Guangming; Ma, Yongmei; Li, Xinhong; Jiang, Lei; Wang, Fosong

2006-03-01

A conductive polymer polyaniline (PANI) was employed to achieve surfaces of both super-hydrophobic and conductive on NaOH etched porous anodic alumina (PAA) membranes. The surfaces exhibit micro- and nanostructures. In the PANI modified PAA membrane, PANI is mainly emeraldine. After the membrane was immersed in HCl, the content of the protonated nitrogen increased, which increased the conductivity.

Development of Omniphobic Desalination Membranes Using a Charged Electrospun Nanofiber Scaffold.

PubMed

Lee, Jongho; Boo, Chanhee; Ryu, Won-Hee; Taylor, André D; Elimelech, Menachem

2016-05-04

In this study, we present a facile and scalable approach to fabricate omniphobic nanofiber membranes by constructing multilevel re-entrant structures with low surface energy. We first prepared positively charged nanofiber mats by electrospinning a blend polymer-surfactant solution of poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) and cationic surfactant (benzyltriethylammonium). Negatively charged silica nanoparticles (SiNPs) were grafted on the positively charged electrospun nanofibers via dip-coating to achieve multilevel re-entrant structures. Grafted SiNPs were then coated with fluoroalkylsilane to lower the surface energy of the membrane. The fabricated membrane showed excellent omniphobicity, as demonstrated by its wetting resistance to various low surface tension liquids, including ethanol with a surface tension of 22.1 mN/m. As a promising application, the prepared omniphobic membrane was tested in direct contact membrane distillation to extract water from highly saline feed solutions containing low surface tension substances, mimicking emerging industrial wastewaters (e.g., from shale gas production). While a control hydrophobic PVDF-HFP nanofiber membrane failed in the desalination/separation process due to low wetting resistance, our fabricated omniphobic membrane exhibited a stable desalination performance for 8 h of operation, successfully demonstrating clean water production from the low surface tension feedwater.

Origin of 1/f noise in hydration dynamics on lipid membrane surfaces

PubMed Central

Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

2015-01-01

Water molecules on lipid membrane surfaces are known to contribute to membrane stability by connecting lipid molecules and acting as a water bridge. Although water structures and diffusivities near the membrane surfaces have been extensively studied, hydration dynamics on the surfaces has remained an open question. Here we investigate residence time statistics of water molecules on the surface of lipid membranes using all-atom molecular dynamics simulations. We show that hydration dynamics on the lipid membranes exhibits 1/f noise. Constructing a dichotomous process for the hydration dynamics, we find that residence times in each state follow a power-law with exponential cutoff and that the process can be regarded as a correlated renewal process where interoccurrence times are correlated. The results imply that the origin of the 1/f noise in hydration dynamics on the membrane surfaces is a combination of a power-law distribution with cutoff of interoccurrence times of switching events and a long-term correlation between the interoccurrence times. These results suggest that the 1/f noise attributed to the correlated renewal process may contribute to the stability of the hydration layers and lipid membranes. PMID:25743377

Effect of pro-inflammatory mediators on membrane-associated mucins expressed by human ocular surface epithelial cells.

PubMed

Albertsmeyer, Ann-Christin; Kakkassery, Vinodh; Spurr-Michaud, Sandra; Beeks, Olivia; Gipson, Ilene K

2010-03-01

Membrane-associated mucins are altered on the ocular surface in non-Sjögren's dry eye. This study sought to determine if inflammatory mediators, present in tears of dry eye patients, regulate membrane-associated mucins MUC1 and -16 at the level of gene expression, protein biosynthesis and/or ectodomain release. A human corneal limbal epithelial cell line (HCLE), which produces membrane-associated mucins, was used. Cells were treated with interleukin (IL)-6, -8, or -17, tumor necrosis factor-alpha (TNF-alpha), and Interferon-gamma (IFN-gamma), or a combination of TNF-alpha and IFN-gamma, or IFN-gamma and IL-17, for 1, 6, 24, or 48 h. Presence of receptors for these mediators was verified by RT-PCR. Effects of the cytokines on expression levels of MUC1 and -16 were determined by real-time PCR, and on mucin protein biosynthesis and ectodomain release in cell lysates and culture media, respectively, by immunoblot analysis. TNF-alpha and IFN-gamma each significantly induced MUC1 expression, cellular protein content and ectodomain release over time. Combined treatment with the two cytokines was not additive. By comparison, one of the inflammatory mediators, IFN-gamma, affected all three parameters-gene expression, cellular protein, and ectodomain release-for MUC16. Combined treatment with TNF-alpha and IFN-gamma showed effects similar to IFN-gamma alone, except that ectodomain release followed that of TNF-alpha, which induced MUC16 ectodomain release. In conclusion, inflammatory mediators present in tears of dry eye patients can affect MUC1 and -16 on corneal epithelial cells and may be responsible for alterations of surface mucins in dry eye.

Vitamin D receptor is present on the neuronal plasma membrane and is co-localized with amyloid precursor protein, ADAM10 or Nicastrin.

PubMed

Dursun, Erdinç; Gezen-Ak, Duygu

2017-01-01

Our recent study indicated that vitamin D and its receptors are important parts of the amyloid processing pathway in neurons. Yet the role of vitamin D receptor (VDR) in amyloid pathogenesis is complex and all regulations over the production of amyloid beta cannot be explained solely with the transcriptional regulatory properties of VDR. Given that we hypothesized that VDR might exist on the neuronal plasma membrane in close proximity with amyloid precursor protein (APP) and secretase complexes. The present study primarily focused on the localization of VDR in neurons and its interaction with amyloid pathology-related proteins. The localization of VDR on neuronal membranes and its co-localization with target proteins were investigated with cell surface staining followed by immunofluorescence labelling. The FpClass was used for protein-protein interaction prediction. Our results demonstrated the localization of VDR on the neuronal plasma membrane and the co-localization of VDR and APP or ADAM10 or Nicastrin and limited co-localization of VDR and PS1. E-cadherin interaction with APP or the γ-secretase complex may involve NOTCH1, NUMB, or FHL2, according to FpClass. This suggested complex might also include VDR, which greatly contributes to Ca+2 hemostasis with its ligand vitamin D. Consequently, we suggested that VDR might be a member of this complex also with its own non-genomic action and that it can regulate the APP processing pathway in this way in neurons.

Phosphatidylinositol 3-phosphates-at the interface between cell signalling and membrane traffic.

PubMed

Marat, Andrea L; Haucke, Volker

2016-03-15

Phosphoinositides (PIs) form a minor class of phospholipids with crucial functions in cell physiology, ranging from cell signalling and motility to a role as signposts of compartmental membrane identity. Phosphatidylinositol 3-phosphates are present at the plasma membrane and within the endolysosomal system, where they serve as key regulators of both cell signalling and of intracellular membrane traffic. Here, we provide an overview of the metabolic pathways that regulate cellular synthesis of PI 3-phosphates at distinct intracellular sites and discuss the mechanisms by which these lipids regulate cell signalling and membrane traffic. Finally, we provide a framework for how PI 3-phosphate metabolism is integrated into the cellular network. © 2016 The Authors.

Ionic protein-lipid interaction at the plasma membrane: what can the charge do?

PubMed

Li, Lunyi; Shi, Xiaoshan; Guo, Xingdong; Li, Hua; Xu, Chenqi

2014-03-01

Phospholipids are the major components of cell membranes, but they have functional roles beyond forming lipid bilayers. In particular, acidic phospholipids form microdomains in the plasma membrane and can ionically interact with proteins via polybasic sequences, which can have functional consequences for the protein. The list of proteins regulated by ionic protein-lipid interaction has been quickly expanding, and now includes membrane proteins, cytoplasmic soluble proteins, and viral proteins. Here we review how acidic phospholipids in the plasma membrane regulate protein structure and function via ionic interactions, and how Ca(2+) regulates ionic protein-lipid interactions via direct and indirect mechanisms. Copyright © 2014 Elsevier Ltd. All rights reserved.

Electron transport and light-harvesting switches in cyanobacteria

PubMed Central

Mullineaux, Conrad W.

2014-01-01

Cyanobacteria possess multiple mechanisms for regulating the pathways of photosynthetic and respiratory electron transport. Electron transport may be regulated indirectly by controlling the transfer of excitation energy from the light-harvesting complexes, or it may be more directly regulated by controlling the stoichiometry, localization, and interactions of photosynthetic and respiratory electron transport complexes. Regulation of the extent of linear vs. cyclic electron transport is particularly important for controlling the redox balance of the cell. This review discusses what is known of the regulatory mechanisms and the timescales on which they occur, with particular regard to the structural reorganization needed and the constraints imposed by the limited mobility of membrane-integral proteins in the crowded thylakoid membrane. Switching mechanisms requiring substantial movement of integral thylakoid membrane proteins occur on slower timescales than those that require the movement only of cytoplasmic or extrinsic membrane proteins. This difference is probably due to the restricted diffusion of membrane-integral proteins. Multiple switching mechanisms may be needed to regulate electron transport on different timescales. PMID:24478787

A phosphoinositide-binding cluster in cavin1 acts as a molecular sensor for cavin1 degradation

PubMed Central

Tillu, Vikas A.; Kovtun, Oleksiy; McMahon, Kerrie-Ann; Collins, Brett M.; Parton, Robert G.

2015-01-01

Caveolae are abundant surface organelles implicated in a range of cellular processes. Two classes of proteins work together to generate caveolae: integral membrane proteins termed caveolins and cytoplasmic coat proteins called cavins. Caveolae respond to membrane stress by releasing cavins into the cytosol. A crucial aspect of this model is tight regulation of cytosolic pools of cavin under resting conditions. We now show that a recently identified region of cavin1 that can bind phosphoinositide (PI) lipids is also a major site of ubiquitylation. Ubiquitylation of lysines within this site leads to rapid proteasomal degradation. In cells that lack caveolins and caveolae, cavin1 is cytosolic and rapidly degraded as compared with cells in which cavin1 is associated with caveolae. Membrane stretching causes caveolar disassembly, release of cavin complexes into the cytosol, and increased proteasomal degradation of wild-type cavin1 but not mutant cavin1 lacking the major ubiquitylation site. Release of cavin1 from caveolae thus leads to exposure of key lysine residues in the PI-binding region, acting as a trigger for cavin1 ubiquitylation and down-regulation. This mutually exclusive PI-binding/ubiquitylation mechanism may help maintain low levels of cytosolic cavin1 in resting cells, a prerequisite for cavins acting as signaling modules following release from caveolae. PMID:26269585

Membrane Bending by Protein Crowding

NASA Astrophysics Data System (ADS)

Stachowiak, Jeanne

2014-03-01

From endosomes and synaptic vesicles to the cristae of the mitochondria and the annulus of the nuclear pore, highly curved membranes are fundamental to the structure and physiology of living cells. The established view is that specific families of proteins are able to bend membranes by binding to them. For example, inherently curved proteins are thought to impose their structure on the membrane surface, while membrane-binding proteins with hydrophobic motifs are thought to insert into the membrane like wedges, driving curvature. However, computational models have recently revealed that these mechanisms would require specialized membrane-bending proteins to occupy nearly 100% of a curved membrane surface, an improbable physiological situation given the immense density and diversity of membrane-bound proteins, and the low expression levels of these specialized proteins within curved regions of the membrane. How then does curvature arise within the complex and crowded environment of cellular membranes? Our recent work using proteins involved in clathrin-mediated endocytosis, as well as engineered protein-lipid interactions, has suggested a new hypothesis - that lateral pressure generated by collisions between membrane-bound proteins can drive membrane bending. Specifically, by correlating membrane bending with quantitative optical measurements of protein density on synthetic membrane surfaces and simple physical models of collisions among membrane-bound proteins, we have demonstrated that protein-protein steric interactions can drive membrane curvature. These findings suggest that a simple imbalance in the concentration of membrane-bound proteins across a membrane surface can drive a membrane to bend, providing an efficient mechanism by which essentially any protein can contribute to shaping membranes.

Rubber particle proteins, HbREF and HbSRPP, show different interactions with model membranes.

PubMed

Berthelot, Karine; Lecomte, Sophie; Estevez, Yannick; Zhendre, Vanessa; Henry, Sarah; Thévenot, Julie; Dufourc, Erick J; Alves, Isabel D; Peruch, Frédéric

2014-01-01

The biomembrane surrounding rubber particles from the hevea latex is well known for its content of numerous allergen proteins. HbREF (Hevb1) and HbSRPP (Hevb3) are major components, linked on rubber particles, and they have been shown to be involved in rubber synthesis or quality (mass regulation), but their exact function is still to be determined. In this study we highlighted the different modes of interactions of both recombinant proteins with various membrane models (lipid monolayers, liposomes or supported bilayers, and multilamellar vesicles) to mimic the latex particle membrane. We combined various biophysical methods (polarization-modulation-infrared reflection-adsorption spectroscopy (PM-IRRAS)/ellipsometry, attenuated-total reflectance Fourier-transform infrared (ATR-FTIR), solid-state nuclear magnetic resonance (NMR), plasmon waveguide resonance (PWR), fluorescence spectroscopy) to elucidate their interactions. Small rubber particle protein (SRPP) shows less affinity than rubber elongation factor (REF) for the membranes but displays a kind of "covering" effect on the lipid headgroups without disturbing the membrane integrity. Its structure is conserved in the presence of lipids. Contrarily, REF demonstrates higher membrane affinity with changes in its aggregation properties, the amyloid nature of REF, which we previously reported, is not favored in the presence of lipids. REF binds and inserts into membranes. The membrane integrity is highly perturbed, and we suspect that REF is even able to remove lipids from the membrane leading to the formation of mixed micelles. These two homologous proteins show affinity to all membrane models tested but neatly differ in their interacting features. This could imply differential roles on the surface of rubber particles. © 2013.

Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid peroxidation in Escherichia coli.

PubMed

Joshi, Suresh G; Cooper, Moogega; Yost, Adam; Paff, Michelle; Ercan, Utku K; Fridman, Gregory; Friedman, Gary; Fridman, Alexander; Brooks, Ari D

2011-03-01

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria.

Nonthermal Dielectric-Barrier Discharge Plasma-Induced Inactivation Involves Oxidative DNA Damage and Membrane Lipid Peroxidation in Escherichia coli▿

PubMed Central

Joshi, Suresh G.; Cooper, Moogega; Yost, Adam; Paff, Michelle; Ercan, Utku K.; Fridman, Gregory; Friedman, Gary; Fridman, Alexander; Brooks, Ari D.

2011-01-01

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria. PMID:21199923

Assembly of the MHC I peptide-loading complex determined by a conserved ionic lock-switch

PubMed Central

Blees, Andreas; Reichel, Katrin; Trowitzsch, Simon; Fisette, Olivier; Bock, Christoph; Abele, Rupert; Hummer, Gerhard; Schäfer, Lars V.; Tampé, Robert

2015-01-01

Salt bridges in lipid bilayers play a decisive role in the dynamic assembly and downstream signaling of the natural killer and T-cell receptors. Here, we describe the identification of an inter-subunit salt bridge in the membrane within yet another key component of the immune system, the peptide-loading complex (PLC). The PLC regulates cell surface presentation of self-antigens and antigenic peptides via molecules of the major histocompatibility complex class I. We demonstrate that a single salt bridge in the membrane between the transporter associated with antigen processing TAP and the MHC I-specific chaperone tapasin is essential for the assembly of the PLC and for efficient MHC I antigen presentation. Molecular modeling and all-atom molecular dynamics simulations suggest an ionic lock-switch mechanism for the binding of TAP to tapasin, in which an unfavorable uncompensated charge in the ER-membrane is prevented through complex formation. Our findings not only deepen the understanding of the interaction network within the PLC, but also provide evidence for a general interaction principle of dynamic multiprotein membrane complexes in immunity. PMID:26611325

Sub-cellular distribution and translocation of TRP channels.

PubMed

Toro, Carlos A; Arias, Luis A; Brauchi, Sebastian

2011-01-01

Cellular electrical activity is the result of a highly complex processes that involve the activation of ion channel proteins. Ion channels make pores on cell membranes that rapidly transit between conductive and non-conductive states, allowing different ions to flow down their electrochemical gradients across cell membranes. In the case of neuronal cells, ion channel activity orchestrates action potentials traveling through axons, enabling electrical communication between cells in distant parts of the body. Somatic sensation -our ability to feel touch, temperature and noxious stimuli- require ion channels able to sense and respond to our peripheral environment. Sensory integration involves the summing of various environmental cues and their conversion into electrical signals. Members of the Transient Receptor Potential (TRP) family of ion channels have emerged as important mediators of both cellular sensing and sensory integration. The regulation of the spatial and temporal distribution of membrane receptors is recognized as an important mechanism for controlling the magnitude of the cellular response and the time scale on which cellular signaling occurs. Several studies have shown that this mechanism is also used by TRP channels to modulate cellular response and ultimately fulfill their physiological function as sensors. However, the inner-working of this mode of control for TRP channels remains poorly understood. The question of whether TRPs intrinsically regulate their own vesicular trafficking or weather the dynamic regulation of TRP channel residence on the cell surface is caused by extrinsic changes in the rates of vesicle insertion or retrieval remain open. This review will examine the evidence that sub-cellular redistribution of TRP channels plays an important role in regulating their activity and explore the mechanisms that control the trafficking of vesicles containing TRP channels.

PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

PubMed

Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

2001-09-18

It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

Surface modification of PTMSP membranes by plasma treatment: Asymmetry of transport in organic solvent nanofiltration.

PubMed

Volkov, A V; Tsarkov, S E; Gilman, A B; Khotimsky, V S; Roldughin, V I; Volkov, V V

2015-08-01

For the first time, the effect of asymmetry of the membrane transport was studied for organic solvents and solutes upon their nanofiltration through the plasma-modified membranes based on poly(1-trimethylsilyl-1-propyne) (PTMSP). Plasma treatment is shown to provide a marked hydrophilization of the hydrophobic PTMSP surface (the contact angle of water decreases from 88 down to 20°) and leads to the development of a negative charge of -5.2 nC/cm(2). The XPS measurements prove the formation of the oxygen-containing groups (Si-O and C-O) due to the surface modification. The AFM images show that the small-scale surface roughness of the plasma-treated PTMSP sample is reduced but the large-scale surface heterogeneities become more pronounced. The modified membranes retain their hydrophilic surface properties even after the nanofiltration tests and 30-day storage under ambient conditions. The results of the filtration tests show that when the membrane is oriented so that its modified layer contacts the feed solution, the membrane permeability for linear alcohols (methanol-propanol) and acetone decreases nearly two times. When the modified membrane surface faces the permeate, the membrane is seen to regain its transport characteristics: the flux becomes equal to that of the unmodified PTMSP. The well-pronounced effect of the transport asymmetry is observed for the solution of the neutral dye Solvent Blue 35 in methanol, ethanol, and acetone. For example, the initial membrane shows the negative retention for the Solvent Blue 35 dye (-16%) upon its filtration from the ethanol solution whereas, for the modified PTMSP membrane, the retention increases up to 17%. Various effects contributing to the asymmetry of the membrane transport characteristics are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

Amphotericin B channels in phospholipid membrane-coated nanoporous silicon surfaces: implications for photovoltaic driving of ions across membranes.

PubMed

Yilma, Solomon; Liu, Nangou; Samoylov, Alexander; Lo, Ting; Brinker, C Jeffrey; Vodyanoy, Vitaly

2007-03-15

The antimycotic agent amphotericin B (AmB) functions by forming complexes with sterols to form ion channels that cause membrane leakage. When AmB and cholesterol mixed at 2:1 ratio were incorporated into phospholipid bilayer membranes formed on the tip of patch pipettes, ion channel current fluctuations with characteristic open and closed states were observed. These channels were also functional in phospholipid membranes formed on nanoporous silicon surfaces. Electrophysiological studies of AmB-cholesterol mixtures that were incorporated into phospholipid membranes formed on the surface of nanoporous (6.5 nm pore diameter) silicon plates revealed large conductance ion channels ( approximately 300 pS) with distinct open and closed states. Currents through the AmB-cholesterol channels on nanoporous silicon surfaces can be driven by voltage applied via conventional electrical circuits or by photovoltaic electrical potential entirely generated when the nanoporous silicon surface is illuminated with a narrow laser beam. Electrical recordings made during laser illumination of AmB-cholesterol containing membrane-coated nanoporous silicon surfaces revealed very large conductance ion channels with distinct open and closed states. Our findings indicate that nanoporous silicon surfaces can serve as mediums for ion-channel-based biosensors. The photovoltaic properties of nanoporous silicon surfaces show great promise for making such biosensors addressable via optical technologies.

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor signaling.

PubMed

Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-09-01

Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Ketamine-induced inhibition of glycogen synthase kinase-3 contributes to the augmentation of AMPA receptor signaling

PubMed Central

Beurel, Eléonore; Grieco, Steven F; Amadei, Celeste; Downey, Kimberlee; Jope, Richard S

2016-01-01

Objectives Sub-anesthetic doses of ketamine have been found to provide rapid antidepressant actions, indicating that the cellular signaling systems targeted by ketamine are potential sites for therapeutic intervention. Ketamine acts as an antagonist of N-methyl-D-aspartate (NMDA) receptors, and animal studies indicate that subsequent augmentation of signaling by α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors is critical for the antidepressant outcome. Methods In this study, we tested if the inhibitory effect of ketamine on glycogen synthase kinase-3 (GSK3) affected hippocampal cell-surface AMPA receptors using immunoblotting of membrane and synaptosomal extracts from wild-type and GSK3 knockin mice. Results Treatment with an antidepressant dose of ketamine increased the hippocampal membrane level of the AMPA glutamate receptor (GluA)1 subunit, but did not alter the localization of GluA2, GluA3, or GluA4. This effect of ketamine was abrogated in GSK3 knockin mice expressing mutant GSK3 that cannot be inhibited by ketamine, demonstrating that ketamine-induced inhibition of GSK3 is necessary for up-regulation of cell surface AMPA GluA1 subunits. AMPA receptor trafficking is regulated by post-synaptic density-95 (PSD-95), a substrate for GSK3. Ketamine treatment decreased the hippocampal membrane level of phosphorylated PSD-95 on Thr-19, the target of GSK3 that promotes AMPA receptor internalization. Conclusions These results demonstrate that ketamine-induced inhibition of GSK3 causes reduced phosphorylation of PSD-95, diminishing the internalization of AMPA GluA1 subunits to allow for augmented signaling through AMPA receptors following ketamine treatment. PMID:27687706

Regulation of basal and reserve cardiac pacemaker function by interactions of cAMP mediated PKA-dependent Ca2+ cycling with surface membrane channels

PubMed Central

Vinogradova, Tatiana M.; Lakatta, Edward G.

2009-01-01

Decades of intensive research of primary cardiac pacemaker, the sinoatrial node, have established potential roles of specific membrane channels in the generation of the diastolic depolarization, the major mechanism allowing sinoatrial node cells generate spontaneous beating. During the last three decades, multiple studies made either in the isolated sinoatrial node or sinoatrial node cells have demonstrated a pivotal role of Ca2+ and, specifically Ca2+-release from sarcoplasmic reticulum, for spontaneous beating of cardiac pacemaker. Recently, spontaneous, rhythmic local subsarcolemmal Ca2+ releases from ryanodine receptors during late half of the diastolic depolarization have been implicated as a vital factor in the generation of sinoatrial node cells spontaneous firing. Local Ca2+ releases are driven by a unique combination of high basal cAMP production by adenylyl cyclases, high basal cAMP degradation by phosphodiesterases and a high level of cAMP-mediated PKA-dependent phosphorylation. These local Ca2+ releases activate an inward Na+-Ca2+ exchange current which accelerates the terminal diastolic depolarization rate and, thus, controls the spontaneous pacemaker firing. Both the basal primary pacemaker beating rate and its modulation via β-adrenergic receptor stimulation appear to be critically dependent upon intact RyR function and local subsarcolemmal sarcoplasmic reticulum generated Ca2+ releases. This review aspires to integrate the traditional viewpoint that has emphasized the supremacy of the ensemble of surface membrane ion channels in spontaneous firing of the primary cardiac pacemaker, and these novel perspectives of cAMP-mediated PKA-dependent Ca2+ cycling in regulation of the heart pacemaker clock, both in the basal state and during β-adrenergic receptor stimulation. PMID:19573534

Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes.

PubMed

Park, Dayoung; Brune, Kristin A; Mitra, Anupam; Marusina, Alina I; Maverakis, Emanual; Lebrilla, Carlito B

2015-11-01

Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Egg-in-Cube: Design and Fabrication of a Novel Artificial Eggshell with Functionalized Surface

PubMed Central

Huang, Wenjing; Arai, Fumihito; Kawahara, Tomohiro

2015-01-01

An eggshell is a porous microstructure that regulates the passage of gases to allow respiration. The chick embryo and its circulatory system enclosed by the eggshell has become an important model for biomedical research such as the control of angiogenesis, cancer therapy, and drug delivery test, because the use of embryo is ethically acceptable and it is inexpensive and small. However, chick embryo and extra-embryonic blood vessels cannot be accessed freely and has poor observability because the eggshell is tough and cannot be seen through, which limits its application. In this study, a novel artificial eggshell with functionalized surface is proposed, which allows the total amount of oxygen to pass into the egg for the chick embryo culturing and has high observability and accessibility for embryo manipulation. First, a 40-mm enclosed cubic-shaped eggshell consisting of a membrane structure and a rigid frame structure is designed, and then the threshold of the membrane thickness suitable for the embryo survival is figured out according to the oxygen-permeability of the membrane structure. The designed artificial eggshell was actually fabricated by using polydimethylsiloxane (PDMS) and polycarbonate (PC) in the current study. Using the fabricated eggshell, chick embryo and extra-embryonic blood vessels can be observed from multiple directions. To test the effectiveness of the design, the cubic eggshells were used to culture chick embryos and survivability was confirmed when PDMS membranes with adequate oxygen permeability were used. Since the surface of the eggshell is transparent, chick embryo tissue development could be observed during the culture period. Additionally, the chick embryo tissues could be accessed and manipulated from outside the cubic eggshell, by using mechanical tools without breakage of the eggshell. The proposed “Egg-in-Cube” with functionalized surface has great potential to serve as a promising platform for biomedical research. PMID:25768929

Probing the Interaction of Dielectric Nanoparticles with Supported Lipid Membrane Coatings on Nanoplasmonic Arrays

PubMed Central

Ferhan, Abdul Rahim; Ma, Gamaliel Junren; Jackman, Joshua A.; Sut, Tun Naw; Park, Jae Hyeon; Cho, Nam-Joon

2017-01-01

The integration of supported lipid membranes with surface-based nanoplasmonic arrays provides a powerful sensing approach to investigate biointerfacial phenomena at membrane interfaces. While a growing number of lipid vesicles, protein, and nucleic acid systems have been explored with nanoplasmonic sensors, there has been only very limited investigation of the interactions between solution-phase nanomaterials and supported lipid membranes. Herein, we established a surface-based localized surface plasmon resonance (LSPR) sensing platform for probing the interaction of dielectric nanoparticles with supported lipid bilayer (SLB)-coated, plasmonic nanodisk arrays. A key emphasis was placed on controlling membrane functionality by tuning the membrane surface charge vis-à-vis lipid composition. The optical sensing properties of the bare and SLB-coated sensor surfaces were quantitatively compared, and provided an experimental approach to evaluate nanoparticle–membrane interactions across different SLB platforms. While the interaction of negatively-charged silica nanoparticles (SiNPs) with a zwitterionic SLB resulted in monotonic adsorption, a stronger interaction with a positively-charged SLB resulted in adsorption and lipid transfer from the SLB to the SiNP surface, in turn influencing the LSPR measurement responses based on the changing spatial proximity of transferred lipids relative to the sensor surface. Precoating SiNPs with bovine serum albumin (BSA) suppressed lipid transfer, resulting in monotonic adsorption onto both zwitterionic and positively-charged SLBs. Collectively, our findings contribute a quantitative understanding of how supported lipid membrane coatings influence the sensing performance of nanoplasmonic arrays, and demonstrate how the high surface sensitivity of nanoplasmonic sensors is well-suited for detecting the complex interactions between nanoparticles and lipid membranes. PMID:28644423

In vitro tympanic membrane position identification with a co-axial fiber-optic otoscope

NASA Astrophysics Data System (ADS)

Sundberg, Mikael; Peebo, Markus; Strömberg, Tomas

2011-09-01

Otitis media diagnosis can be assisted by measuring the shape of the tympanic membrane. We have developed an ear speculum for an otoscope, including spatially distributed source and detector optical fibers, to generate source-detector intensity matrices (SDIMs), representing the curvature of surfaces. The surfaces measured were a model ear with a latex membrane and harvested temporal bones including intact tympanic membranes. The position of the tympanic membrane was shifted from retracted to bulging by air pressure and that of the latex membrane by water displacement. The SDIM was normalized utilizing both external (a sheared flat plastic cylinder) and internal references (neutral position of the membrane). Data was fitted to a two-dimensional Gaussian surface representing the shape by its amplitude and offset. Retracted and bulging surfaces were discriminated for the model ear by the sign of the Gaussian amplitude for both internal and external reference normalization. Tympanic membranes were separated after a two-step normalization: first to an external reference, adjusted for the distance between speculum and the surfaces, and second by comparison with an average normally positioned SDIM from tympanic membranes. In conclusion, we have shown that the modified otoscope can discriminate between bulging and retracted tympanic membranes in a single measurement, given a two-step normalization.

Membrane projection lithography

DOEpatents

Burckel, David Bruce; Davids, Paul S; Resnick, Paul J; Draper, Bruce L

2015-03-17

The various technologies presented herein relate to a three dimensional manufacturing technique for application with semiconductor technologies. A membrane layer can be formed over a cavity. An opening can be formed in the membrane such that the membrane can act as a mask layer to the underlying wall surfaces and bottom surface of the cavity. A beam to facilitate an operation comprising any of implantation, etching or deposition can be directed through the opening onto the underlying surface, with the opening acting as a mask to control the area of the underlying surfaces on which any of implantation occurs, material is removed, and/or material is deposited. The membrane can be removed, a new membrane placed over the cavity and a new opening formed to facilitate another implantation, etching, or deposition operation. By changing the direction of the beam different wall/bottom surfaces can be utilized to form a plurality of structures.

Effect of gamma-ray irradiation at low doses on the performance of PES ultrafiltration membrane

NASA Astrophysics Data System (ADS)

Zhang, Xue; Niu, Lixia; Li, Fuzhi; Yu, Suping; Zhao, Xuan; Hu, Hongying

2016-10-01

The influence of gamma irradiation on the performance of polyether sulfone (PES) ultrafiltration (UF) membrane was investigated at low absorbed doses (0-75 kGy) using a cobalt source. The performance of the UF membranes was tested using low level radioactive wastewater (LLRW) containing three types of surfactants (anionic, cationic and nonionic surfactants). The physical and chemical properties of membrane surface were analyzed, and relationships between these properties and separation performance and fouling characteristics were determined. At 10-75 kGy irradiation, there were no significant changes observed in the membrane surface roughness or polymer functional groups, however the contact angle decreased sharply from 92° to ca. 70° at irradiation levels as low as 10 kGy. When membranes were exposed to the surfactant-containing LLRW, the flux decreased more sharply for higher dosed irradiated membranes, while flux in virgin membranes increased during the filtration processes. The study highlights that fouling properties of membrane may be changed due to the changes of surface hydrophilicity at low dose irradiation, while other surface properties and retentions remain stable. Therefore, a membrane fouling test with real or simulated wastewater is recommended to fully evaluate the membrane irradiation resistance.

Study on surface adhesion of Plasma modified Polytetrafluoroethylene hollow fiber membrane

NASA Astrophysics Data System (ADS)

Chen, Jiangrong; Zhang, Huifeng; Liu, Guochang; Guo, Chungang; Lv, Jinglie; Zhangb, Yushan

2018-01-01

Polytetrafluoroethylene (PTFE) is popular membrane material because of its excellent thermal stability, chemical stability and mechanical stability. However, the low surface energy and non-sticky property of PTFE present challenges for modification. In the present study, plasma treatment was performed to improve the surface adhesion of PTFE hollow fiber membrane. The effect of discharge voltage, treatment time on the adhesion of PTFE hollow fiber membrane was symmetrically evaluated. Results showed that the plasma treatment method contributed to improve the surface activity and roughness of PTFE hollow fiber membrane, and the adhesion strength depend significantly on discharge voltage, which was beneficial to seepage pressure of PTFE hollow fiber membrane module. The adhesion strength of PTFE membrane by plasma treated at 220V for 3min reached as high as 86.2 N, far surpassing the adhesion strength 12.7 N of pristine membrane. Furthermore, improvement of content of free radical and composition analysis changes of the plasma modified PTFE membrane were investigated. The seepage pressure of PTFE membrane by plasma treated at 220V for 3min was 0.375 MPa, which means that the plasma treatment is an effective technique to improve the adhesion strength of membrane.

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Jaemin, E-mail: jmj1103@kirams.re.kr; Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, Seoul; Conboy, Irina M., E-mail: iconboy@berkeley.edu

2011-10-14

Highlights: {yields} PS broadly and persistently trans-locates to the outer leaflet of plasma membrane during myoblast fusion into myotubes. {yields} Robust myotubes are formed when PS liposomes are added exogenously. {yields} PS increases the width of de novo myotubes and the numbers of myonuclei, but not the myotube length. {yields} Annexin V or PS antibody inhibits myotube formation by masking exposed PS. -- Abstract: Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generallymore » but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell-cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.« less

Fabrication of polyamide thin-film nanocomposite membranes with enhanced surface charge for nitrate ion removal from water resources.

PubMed

Ghaee, A; Zerafat, M M; Askari, P; Sabbaghi, S; Sadatnia, B

2017-03-01

Exclusion due to membrane surface charge is considered as one of the main separation mechanisms occurring in charged membranes, which can be varied through various approaches to affect membrane rejection performance. In this study, thin-film composite (TFC) polyamide (PA) membranes were fabricated via interfacial polymerization of m-phenylenediamine (m-PDA) and 2,4-diaminobenzene sulfonic acid with trimesoyl chloride (TMC) on a polysulfone sub-layer. The ability of the prepared membrane to remove nitrate ions from water resources has been investigated. In order to improve membrane permeability, zeolite-PA thin film nanocomposite (TFN) membranes were fabricated by incorporating natural zeolite nanoparticles obtained through ball milling of an Iranian natural zeolite powder in the interfacial polymerization process. The size, morphology and specific surface area of the as-obtained nanozeolite were characterized using particle size analysis, FE-SEM and BET. The functional groups, morphology and surface charge of the membrane were characterized using ATR-FTIR, SEM and zeta potential analyses. Also, field-emission scanning electron microscopy (FE-SEM) and energy dispersive X-ray spectroscopy (EDS) were used to determine the distribution of nanozeolite in TFN membranes. The influence of zeolite addition to surface roughness was accessed by atomic force microscopy. The performance of TFC and TFN membranes was evaluated in terms of pure water flux and nitrate rejection. The results showed that in case of sulfonated diamine, nitrate ions rejection was enhanced from 63% to 85% which could be attributed to surface charge enhancement. TFN permeability was almost doubled by the addition of nanozeolite.

Fluorescent Probe Study of AOT Vesicle Membranes and Their Alteration upon Addition of Aniline or the Aniline Dimer p-Aminodiphenylamine (PADPA).

PubMed

Iwasaki, Fumihiko; Luginbühl, Sandra; Suga, Keishi; Walde, Peter; Umakoshi, Hiroshi

2017-02-28

Artificial vesicles formed from sodium bis(2-ethylhexyl) sulfosuccinate (AOT) in aqueous solution are used successfully as additives for enzymatic oligomerizations or polymerizations of aniline or the aniline dimer p-aminodiphenylamine (PADPA) under slightly acidic conditions (e.g., pH 4.3 with horseradish peroxidase and hydrogen peroxide as oxidants). In these systems, the reactions occur membrane surface-confined. Therefore, (i) the physicochemical properties of the vesicle membrane and (ii) the interaction of aniline or PADPA with the AOT membrane play crucial roles in the progress and final outcome of the reactions. For this reason, the properties of AOT vesicles with and without added aniline or PADPA were investigated by using two fluorescent membrane probes: 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-lauroyl-2-dimethylaminonaphthalene (Laurdan). DPH and Laurdan were used as "sensors" of the membrane fluidity, surface polarity, and membrane phase state. Moreover, the effect of hexanol, alone or in combination with aniline or PADPA, as a possible modifier of the AOT membrane, was also studied with the aim of evaluating whether the membrane fluidity and surface polarity is altered significantly by hexanol, which, in turn, may have an influence on the mentioned types of reactions. The data obtained indicate that the AOT vesicle membrane at room temperature and pH 4.3 (0.1 M NaH 2 PO 4 ) is more fluid and has a more polar surface than in the case of fluid phospholipid vesicle membranes formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Furthermore, the fluorescence measurements indicate that mixed AOT-hexanol membranes are less fluid than pure AOT membranes and that they have a lower surface polarity than pure AOT membranes. PADPA strongly binds to AOT and to mixed AOT/hexanol membranes and leads to drastic changes in the membrane properties (decrease in fluidity and surface polarity), resulting in Laurdan fluorescence spectra, which are characteristic for intramembrane phase separations (coexistence of ordered and disordered domains). This means that highly fluid AOT membranes transform upon the addition of PADPA into membranes that have ordered domains. Although the relevance of this finding for the enzymatic oligomerization of PADPA is not yet clear, it is also of interest if one likes to use heterogeneous vesicle membranes as additives for carrying out membrane surface-confined reactions that do not necessarily involve PADPA as a reactant.

Surface interactions and fouling properties of Micrococcus luteus with microfiltration membranes.

PubMed

Feng, Lei; Li, Xiufen; Song, Ping; Du, Guocheng; Chen, Jian

2011-11-01

This study was conducted to investigate microbial adhesion of Micrococcus luteus to polypropylene (PP) and polyvinylidene fluoride (PVDF) membranes in relation to the variation of the interfacial energies in the membrane-bacteria systems, for revealing effects of short-range surface interactions on filtration behavior. Both the membranes and M. luteus showed typical strong electron donors and hydrophilic properties. The AB component was dominant in the interfacial energies of the two membrane-bacteria systems. M. luteus presented larger negative U(mlb)(XDLVO) to the PP membrane than to the PVDF membrane. The adhesion experiments also proved that M. luteus had higher adhesion percentage to the PP membrane. This study demonstrated that the adhesion potentials of M. luteus to the PP and PVDF membranes might be explained in terms of bacterium, membrane, and intervening medium surface properties, which are mainly determined by the interfacial energies in the systems according to the XDLVO theory.

Grazing incidence x-ray diffraction analysis of zeolite NaA membranes on porous alumina tubes.

PubMed

Kyotani, Tomohiro

2006-07-01

Zeolite NaA-type membranes hydrothermally synthesized on porous alumina tubes, for dehydration process, were characterized by grazing incidence 2 theta scan X-ray diffraction analysis (GIXRD). The fine structure of the membrane was studied fractionally for surface layer and for materials embedded in the porous alumina tube. The thickness of the surface layer on the porous alumina tube in the membranes used in this study was approximately 2-3 microm as determined from transmission electron microscopy with focused ion beam thin-layer specimen preparation technique (FIB-TEM). To discuss the effects of the membrane surface morphology on the GIXRD measurements, CaA-type membrane prepared by ion exchange from the NaA-type membrane and surface-damaged NaA-type membrane prepared by water leaching were also studied. For the original NaA-type membrane, 2 theta scan GIXRD patterns could be clearly measured at X-ray incidence angles (alpha) ranging from 0.1 to 2.0 deg in increments of 0.1 deg. The surface layers of the 2 - 3 microm on the porous alumina tube correspond to the alpha values up to ca. 0.2 deg. For the CaA-type and the surface-damaged NaA-type membranes, however, diffraction patterns from the surface layer could not be successfully detected and the others were somewhat broad. For all the three samples, diffraction intensities of both zeolite and alumina increased with depth (X-ray incidence angle, alpha) in the porous alumina tube region. The depth profile analysis of the membranes based on the GIXRD first revealed that amount of zeolite crystal embedded in the porous alumina tube is much larger than that in the surface layer. Thus, the 2 theta scan GIXRD is a useful method to study zeolite crystal growth mechanism around (both inside and outside) the porous alumina support during hydrothermal synthesis and to study water permeation behavior in the dehydration process.

An intracellular motif of GLUT4 regulates fusion of GLUT4-containing vesicles.

PubMed

Heyward, Catherine A; Pettitt, Trevor R; Leney, Sophie E; Welsh, Gavin I; Tavaré, Jeremy M; Wakelam, Michael J O

2008-05-20

Insulin stimulates glucose uptake by adipocytes through increasing translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. Fusion of GLUT4-containing vesicles at the cell surface is thought to involve phospholipase D activity, generating the signalling lipid phosphatidic acid, although the mechanism of action is not yet clear. Here we report the identification of a putative phosphatidic acid-binding motif in a GLUT4 intracellular loop. Mutation of this motif causes a decrease in the insulin-induced exposure of GLUT4 at the cell surface of 3T3-L1 adipocytes via an effect on vesicle fusion. The potential phosphatidic acid-binding motif identified in this study is unique to GLUT4 among the sugar transporters, therefore this motif may provide a unique mechanism for regulating insulin-induced translocation by phospholipase D signalling.

Preparation of robust braid-reinforced poly(vinyl chloride) ultrafiltration hollow fiber membrane with antifouling surface and application to filtration of activated sludge solution.

PubMed

Zhou, Zhuang; Rajabzadeh, Saeid; Fang, Lifeng; Miyoshi, Taro; Kakihana, Yuriko; Matsuyama, Hideto

2017-08-01

Braid-reinforced hollow fiber membranes with high mechanical properties and considerable antifouling surface were prepared by blending poly(vinyl chloride) (PVC) with poly(vinyl chloride-co-poly(ethylene glycol) methyl ether methacrylate) (poly(VC-co-PEGMA)) copolymer via non-solvent induced phase separation (NIPS). The tensile strength of the braid-reinforced PVC hollow fiber membranes were significantly larger than those of previously reported various types of PVC hollow fiber membranes. The high interfacial bonding strength indicated the good compatibility between the coating materials and the surface of polyethylene terephthalate (PET)-braid. Owing to the surface segregation phenomena, the membrane surface PEGMA coverage increased upon increasing the poly(VC-co-PEGMA)/PVC blending ratio, resulting in higher hydrophilicities and bovine serum albumin (BSA) repulsion. To compare the fouling properties, membranes with similar PWPs were prepared by adjusting the dope solution composition to eliminate the effect of hydrodynamic conditions on the membrane fouling performance. The blend membranes surface exhibited considerable fouling resistance to the molecular adsorption from both BSA solution and activated sludge solution. In both cases, the flux recovered to almost 80% of the initial flux using only water backflush. Considering their great mechanical properties and antifouling resistance to activated sludge solution, these novel membranes show good potential for application in wastewater treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Pore surface fractal analysis of palladium-alumina ceramic membrane using Frenkel-Halsey-Hill (FHH) model.

PubMed

Ahmad, A L; Mustafa, N N N

2006-09-15

The alumina ceramic membrane has been modified by the addition of palladium in order to improve the H(2) permeability and selectivity. Palladium-alumina ceramic membrane was prepared via a sol-gel method and subjected to thermal treatment in the temperature range 500-1100 degrees C. Fractal analysis from nitrogen adsorption isotherm is used to study the pore surface roughness of palladium-alumina ceramic membrane with different chemical composition (nitric acid, PVA and palladium) and calcinations process in terms of surface fractal dimension, D. Frenkel-Halsey-Hill (FHH) model was used to determine the D value of palladium-alumina membrane. Following FHH model, the D value of palladium-alumina membrane increased as the calcinations temperature increased from 500 to 700 degrees C but decreased after calcined at 900 and 1100 degrees C. With increasing palladium concentration from 0.5 g Pd/100 ml H(2)O to 2 g Pd/100 ml H(2)O, D value of membrane decreased, indicating to the smoother surface. Addition of higher amount of PVA and palladium reduced the surface fractal of the membrane due to the heterogeneous distribution of pores. However, the D value increased when nitric acid concentration was increased from 1 to 15 M. The effect of calcinations temperature, PVA ratio, palladium and acid concentration on membrane surface area, pore size and pore distribution also studied.

Amino-functionalized surface modification of polyacrylonitrile hollow fiber-supported polydimethylsiloxane membranes

NASA Astrophysics Data System (ADS)

Hu, Leiqing; Cheng, Jun; Li, Yannan; Liu, Jianzhong; Zhou, Junhu; Cen, Kefa

2017-08-01

This study aimed to improve surface polarity of polydimethylsiloxane (PDMS) membranes and provide surface active sites which were easy to react with other chemicals. 3-Aminopropyltriethoxysilane (APTES) containing an amino group was introduced into a PDMS membrane by crosslinking to prepare polyacrylonitrile hollow fiber-supported PDMS membranes with an amino-functionalized surface. Fourier transform infrared and X-ray photoelectron spectroscopic analyses proved the existence of APTES and its amino group in the PDMS membrane. The concentration of N atoms on the PDMS membrane surface reached ∼6% when the mass ratio of APTES/PDMS oligomer in the PDMS coating solution was increased to 4/3. The water contact angle decreased from ∼114° to ∼87.5°, indicating the improved surface polarization of the PDMS membrane. The density and swelling degree of the PDMS membrane decreased and increased, respectively, with increasing APTES content in PDMS. This phenomenon increased CO2 permeability and decreased CO2/H2 selectivity, CO2/CH4 selectivity, and CO2/N2 selectivity. When the mass ratio of APTES/PDMS oligomer was increased from 0 to 4/3, the CO2 permeation rate of the hollow fiber-supported PDMS membranes initially decreased from ∼2370 GPU to ∼860 GPU and then increased to ∼2000 GPU due to the change in coating solution viscosity.

Construction of Hierarchical Fouling Resistance Surfaces onto Poly(vinylidene fluoride) Membranes for Combating Membrane Biofouling.

PubMed

Li, Xue; Hu, Xuefeng; Cai, Tao

2017-05-09

Owing to the highly hydrophobic nature, fluoropolymer membranes usually suffer from serious fouling problem, and therefore largely limited their practical applications. Also, the development of environmentally benign and nonreleasing antifouling coatings onto the inert fluoropolymer membranes remains a great challenge and is of prime importance for various scientific interests and industrial applications. In the present work, a facile and effective approach for the construction of hierarchical fouling resistance surfaces onto the poly(vinylidene fluoride) (PVDF) membranes was developed. Graft copolymers of PVDF with poly(hyperbranched polyglycerol methacrylamide) side chains (PVDF-g-PHPGMA copolymers) were synthesized via reversible addition-fragmentation chain transfer (RAFT) graft copolymerization of pentafluorophenyl methacrylate (PFMA) with the ozone-preactivated PVDF, followed by activated ester-amine reaction of PPFMA chains with amino-terminated hyperbranched polyglycerol (HPG-NH 2 ). The copolymers could be simply processed into microfiltration (MF) membranes with surface-tethered PHPGMA side chains on the membrane and pore surfaces by nonsolvent induced phase inversion. Furthermore, the PVDF-g-PHPGMA-g-PSBMA membrane was prepared via surface-initiated atom transfer radical polymerization (SI-ATRP) of zwitterionic monomer, N-(3-sulfopropyl)-N-(methacryloxyethyl)-N,N-dimethylammonium betaine (SBMA) from the PVDF-g-PHPGMA membrane and pore surfaces. Arise from a synergistic effect of the dendritic architecture of PHPGMA branches and "superhydrophilic" nature of PSBMA brushes, the PVDF-g-PHPGMA-g-PSBMA membranes exhibit superior resistance to protein and bacteria adhesion with insignificant cytotoxicity effects, making the membranes potentially useful for water treatment and biomedical applications. One may find the present study a general and effective method for the fabrication of antifouling fluoropolymer membranes in a controllable and green manner.

E-cadherin transport from the trans-Golgi network in tubulovesicular carriers is selectively regulated by golgin-97.

PubMed

Lock, John G; Hammond, Luke A; Houghton, Fiona; Gleeson, Paul A; Stow, Jennifer L

2005-12-01

E-cadherin is a cell-cell adhesion protein that is trafficked and delivered to the basolateral cell surface. Membrane-bound carriers for the post-Golgi exocytosis of E-cadherin have not been characterized. Green fluorescent protein (GFP)-tagged E-cadherin (Ecad-GFP) is transported from the trans-Golgi network (TGN) to the recycling endosome on its way to the cell surface in tubulovesicular carriers that resemble TGN tubules labeled by members of the golgin family of tethering proteins. Here, we examine the association of golgins with tubular carriers containing E-cadherin as cargo. Fluorescent GRIP domains from golgin proteins replicate the membrane binding of the full-length proteins and were coexpressed with Ecad-GFP. The GRIP domains of p230/golgin-245 and golgin-97 had overlapping but nonidentical distributions on the TGN; both domains were on TGN-derived tubules but only the golgin-97 GRIP domain coincided with Ecad-GFP tubules in live cells. When the Arl1-binding endogenous golgins, p230/golgin-245 and golgin-97 were displaced from Golgi membranes by overexpression of the p230 GRIP domain, trafficking of Ecad-GFP was inhibited. siRNA knockdown of golgin-97 also inhibited trafficking of Ecad-GFP. Thus, the GRIP domains of p230/golgin-245 and golgin-97 bind discriminately to distinct membrane subdomains of the TGN. Golgin-97 is identified as a selective and essential component of the tubulovesicular carriers transporting E-cadherin out of the TGN.

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

PubMed Central

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

2001-01-01

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

Surface engineering: molecularly imprinted affinity membranes by photograft polymerization

NASA Astrophysics Data System (ADS)

Matuschewski, Heike; Sergeyeva, Tatiana A.; Bendig, Juergen; Piletsky, Sergey A.; Ulbricht, Matthies; Schedler, Uwe

2001-02-01

Commercial polymer microfiltration membranes were surface-modified with a graft copolymer of a functional monomer and a crosslinker in the presence of a template (triazine-herbicide). As result, membranes covered with a thin layer of imprinted polymer (MIP) selective to the template were obtained. The influence of the polymerization conditions on membrane recognition properties was studied by membranes

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

PubMed Central

Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

Two component-three dimensional catalysis

DOEpatents

Schwartz, Michael; White, James H.; Sammells, Anthony F.

2002-01-01

This invention relates to catalytic reactor membranes having a gas-impermeable membrane for transport of oxygen anions. The membrane has an oxidation surface and a reduction surface. The membrane is coated on its oxidation surface with an adherent catalyst layer and is optionally coated on its reduction surface with a catalyst that promotes reduction of an oxygen-containing species (e.g., O.sub.2, NO.sub.2, SO.sub.2, etc.) to generate oxygen anions on the membrane. The reactor has an oxidation zone and a reduction zone separated by the membrane. A component of an oxygen containing gas in the reduction zone is reduced at the membrane and a reduced species in a reactant gas in the oxidation zone of the reactor is oxidized. The reactor optionally contains a three-dimensional catalyst in the oxidation zone. The adherent catalyst layer and the three-dimensional catalyst are selected to promote a desired oxidation reaction, particularly a partial oxidation of a hydrocarbon.

Controlling the morphology and performance of FO membrane via adjusting the atmosphere humidity during casting procedure

NASA Astrophysics Data System (ADS)

Zuo, Hao-Ran; Cao, Gui-Ping; Wang, Meng; Zhang, Huan-Huan; Song, Chen-Chen; Fang, Xu; Wang, Tao

2018-03-01

Forward osmosis (FO) has received great interest for its considerable potential in a wide range of fields. In this work, the morphology and performance of FO membrane were regulated by adjusting the atmosphere humidity (HC) of casting procedure. The polysulfone support layer was casted under various atmosphere humidity levels ranging from 40% to 80%. By multi-techniques such as SEM, AFM, and XPS, it was proved that the atmosphere humidity had modified the surface morphology and thickness of the skin layer in support layer, which contributed up to 90% of the structure parameter, resulting in distinct morphology, thickness, and cross-linking degree of active layer. The active layer with sparse bead-like wrinkles on the smooth surface of support layer casted at HC = 65% showed the highest water permeability [26.9 (L/m2 h MPa)] and considerable low salt permeability [0.0390 (L/m2 h)]. It was found that the water flux of FO-65 was 27% and 46% higher than that of FO-80 in AL-DS and AL-FS mode, respectively, and the salt rejection was as high as 98%. Our work highlighted the importance of considering the effect of atmosphere humidity during casting when design an FO membrane for appropriate performance.

Identification of membrane proteome of Paracoccidioides lutzii and its regulation by zinc

PubMed Central

de Curcio, Juliana Santana; Silva, Marielle Garcia; Silva Bailão, Mirelle Garcia; Báo, Sônia Nair; Casaletti, Luciana; Bailão, Alexandre Mello; de Almeida Soares, Célia Maria

2017-01-01

Aim: During infection development in the host, Paracoccidioides spp. faces the deprivation of micronutrients, a mechanism called nutritional immunity. This condition induces the remodeling of proteins present in different metabolic pathways. Therefore, we attempted to identify membrane proteins and their regulation by zinc in Paracoccidioides lutzii. Materials & methods: Membranes enriched fraction of yeast cells of P. lutzii were isolated, purified and identified by 2D LC–MS/MS detection and database search. Results & conclusion: Zinc deprivation suppressed the expression of membrane proteins such as glycoproteins, those involved in cell wall synthesis and those related to oxidative phosphorylation. This is the first study describing membrane proteins and the effect of zinc deficiency in their regulation in one member of the genus Paracoccidioides. PMID:29134119

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment.

PubMed

Ma, Zhan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Wang, Yingzhi; Yang, Cuixia; Wang, Wenjuan; Du, Yan; Zhou, Muqing; Gao, Feng

2010-08-01

F1Fo-ATP synthase was originally thought to exclusively locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic F1Fo-ATP synthase on the outside of the cell membrane. Ectopic ATP synthase was proposed as a marker for tumor target therapy. Nevertheless, the protein transport mechanism of the ectopic ATP synthase is still unclear. The specificity of the ectopic ATP synthase, with regard to tumors, is questioned because of its widespread expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it into HepG2 cells to study the localization of the ATP synthase. The expression of ATP5B was analyzed in six cell lines with different 'malignancies'. These cells were cultured in both normal and tumor-like acidic and hypoxic conditions. The results suggested that the ectopic expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic activity of ectopic ATP synthase were similar on the surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP synthase was not up-regulated in tumor-like acidic and hypoxic microenvironments. However, the catalytic activity of ectopic ATP synthase was up-regulated in tumor-like microenvironments. Therefore, the specificity of ectopic ATP synthase for tumor target therapy relies on the high level of catalytic activity that is observed in acidic and hypoxic microenvironments in tumor tissues.

Plasma surface modification of nanofiltration (NF) thin-film composite (TFC) membranes to improve anti organic fouling

NASA Astrophysics Data System (ADS)

Kim, Eun-Sik; Yu, Qingsong; Deng, Baolin

2011-09-01

Commercial nanofiltration (NF) thin-film composite (TFC) membranes were treated by low-pressure NH3 plasma, and the effects of the plasma treatment were investigated in terms of the membrane hydrophilicity, pure water flux, salt rejection, protein adsorption, and humic acid fouling. Experimental results indicated that the membrane surface hydrophilicity was increased by the plasma treatment, and changes in the hydrophilicity as well as membrane performance including permeate flux and fouling varied with the original membrane characteristics (e.g., roughness and hydrophilicity). Water flux of plasma treated membranes was the highest with 10 min and 90 W of plasma treatment, and salt rejection was mainly affected by the intensity of the plasma power. Results of bovine serum albumin (BSA) adsorption demonstrated that the protein adsorption decreased with increasing plasma treatment time. The plasma treatment that resulted in more negatively charged surfaces could also better prevent Aldrich humic acid (AHA) attachment on the membrane surface.

Modified polyether-sulfone membrane: a mini review

PubMed Central

Alenazi, Noof A.; Hussein, Mahmoud A.; Alamry, Khalid A.; Asiri, Abdullah M.

2017-01-01

Abstract Polyethersulfone has been widely used as a promising material in medical applications and waste-treatment membranes since it provides excellent mechanical and thermal properties. Hydrophobicity of polyethersulfone is considered one main disadvantage of using this material because hydrophobic surface causes biofouling effects to the membrane which is always thought to be a serious limitation to the use of polyethersulfone in membrane technology. Chemical modification to the material is a promising solution to this problem. More specifically surface modification is an excellent technique to introduce hydrophilic properties and functional groups to the polyethersulfone membrane surface. This review covers chemical modifications of the polyethersulfone and covers different methods used to enhance the hydrophilicity of polyethersulfone membrane. In particular, the addition of amino functional groups to polyethersulfone is used as a fundamental method either to introduce hydrophilic properties or introduce nanomaterials to the surface of polyethersulfone membrane. This work reviews also previous research reports explored the use of amino functionalized polyethersulfone with different nanomaterials to induce biological activity and reduce fouling effects of the fabricated membrane. PMID:29491825

Modified polyether-sulfone membrane: a mini review.

PubMed

Alenazi, Noof A; Hussein, Mahmoud A; Alamry, Khalid A; Asiri, Abdullah M

2017-01-01

Polyethersulfone has been widely used as a promising material in medical applications and waste-treatment membranes since it provides excellent mechanical and thermal properties. Hydrophobicity of polyethersulfone is considered one main disadvantage of using this material because hydrophobic surface causes biofouling effects to the membrane which is always thought to be a serious limitation to the use of polyethersulfone in membrane technology. Chemical modification to the material is a promising solution to this problem. More specifically surface modification is an excellent technique to introduce hydrophilic properties and functional groups to the polyethersulfone membrane surface. This review covers chemical modifications of the polyethersulfone and covers different methods used to enhance the hydrophilicity of polyethersulfone membrane. In particular, the addition of amino functional groups to polyethersulfone is used as a fundamental method either to introduce hydrophilic properties or introduce nanomaterials to the surface of polyethersulfone membrane. This work reviews also previous research reports explored the use of amino functionalized polyethersulfone with different nanomaterials to induce biological activity and reduce fouling effects of the fabricated membrane.

Comparing the antifouling effects of activated carbon and TiO2 in ultrafiltration membrane development.

PubMed

Liu, Qianyu; Huang, Shaobin; Zhang, Yongqing; Zhao, Shuaifei

2018-04-01

We use activated carbon (AC) and titanium oxide (TiO 2 ) nanomaterials as the additives to prepare four polyvinylidene fluoride (PVDF) based ultrafiltration membranes by nonsolvent induced phase separation. The surface properties (pore size, porosity, hydrophilicity and roughness) of the membranes are characterized by scanning electron microscopy, water contact angle measurement, and atomic force microscopy. The chemical properties of the membranes are evaluated by Fourier transform infrared spectroscopy with attenuated total reflection and X-ray diffraction. All these additives can improve the surface hydrophilicity and water permeation flux of the membrane. However, the addition of TiO 2 nanoparticles (20-30 nm) results in larger surface porosities and pore sizes, which causes more severe membrane fouling compared with the neat PVDF membrane. The PVDF-AC membrane exhibits excellent fouling resistance. Particularly, the irreversible fouling after blending AC into PVDF reduces dramatically from 40% to 25%. The antifouling performance of the PVDF-AC membrane may result from the improved hydrophilicity and the favorable surface and structure properties of the membrane. To the best of our knowledge, this is the first demonstration of the antifouling function of AC in membrane preparation. This study suggests that AC could be a new type of nanomaterial for developing antifouling membranes. Copyright © 2018 Elsevier Inc. All rights reserved.

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

PubMed

Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Targeting malaria parasite proteins to the erythrocyte.

PubMed

Templeton, Thomas J; Deitsch, Kirk W

2005-09-01

The intraerythrocytic stages of the protozoan parasite Plasmodium falciparum reside within a parasitophorous vacuole (PV) and set up unique "extraparasite, intraerythrocyte" protein-trafficking pathways that target parasite-encoded proteins to the erythrocyte cytoplasm and cell surface. Two recent articles report the identification of trafficking motifs that regulate the transport of parasite-encoded proteins across the PV. These articles greatly aid the annotation of the parasite "secretome" catalog of proteins that are targeted to the erythrocyte cytoplasm or cell membrane.

Method of separating organic contaminants from fluid feedstreams with polyphosphazene membranes

DOEpatents

McCaffrey, Robert R.; Cummings, Daniel G.

1991-01-01

A method is provided for separating halogenated hydrocarbons from a fluid feedstream. The fluid feedstream is flowed across a first surface of a polyphosphazene semipermeable membrane. At least one halogenated hydrocarbon from the fluid feedstream permeates through the polyphosphazene semipermeable membrane to a second opposed surface of the semipermeable membrane. Then the permeated polar hydrocarbon is removed from the second opposed surface of the polyphosphazene semipermeable membrane. Outstanding and unexpected separation selectivities on the order of 10,000 were obtained for methylene chloride when a methylene chloride in water feedstream was flowed across the polyphosphazene semipermeable membrane in the invented method.

Mitochondrial and Plasma Membrane Pools of Stomatin-Like Protein 2 Coalesce at the Immunological Synapse during T Cell Activation

PubMed Central

Christie, Darah A.; Kirchhof, Mark G.; Vardhana, Santosh; Dustin, Michael L.; Madrenas, Joaquín

2012-01-01

Stomatin-like protein 2 (SLP-2) is a member of the stomatin – prohibitin – flotillin – HflC/K (SPFH) superfamily. Recent evidence indicates that SLP-2 is involved in the organization of cardiolipin-enriched microdomains in mitochondrial membranes and the regulation of mitochondrial biogenesis and function. In T cells, this role translates into enhanced T cell activation. Although the major pool of SLP-2 is associated with mitochondria, we show here that there is an additional pool of SLP-2 associated with the plasma membrane of T cells. Both plasma membrane-associated and mitochondria-associated pools of SLP-2 coalesce at the immunological synapse (IS) upon T cell activation. SLP-2 is not required for formation of IS nor for the re-localization of mitochondria to the IS because SLP-2-deficient T cells showed normal re-localization of these organelles in response to T cell activation. Interestingly, upon T cell activation, we found the surface pool of SLP-2 mostly excluded from the central supramolecular activation complex, and enriched in the peripheral area of the IS where signalling TCR microclusters are located. Based on these results, we propose that SLP-2 facilitates the compartmentalization not only of mitochondrial membranes but also of the plasma membrane into functional microdomains. In this latter location, SLP-2 may facilitate the optimal assembly of TCR signalosome components. Our data also suggest that there may be a net exchange of membrane material between mitochondria and plasma membrane, explaining the presence of some mitochondrial proteins in the plasma membrane. PMID:22623988

Structural features and dynamic investigations of the membrane-bound cytochrome P450 17A1.

PubMed

Cui, Ying-Lu; Xue, Qiao; Zheng, Qing-Chuan; Zhang, Ji-Long; Kong, Chui-Peng; Fan, Jing-Rong; Zhang, Hong-Xing

2015-10-01

Cytochrome P450 (CYP) 17A1 is a dual-function monooxygenase with a critical role in the synthesis of many human steroid hormones. The enzyme is an important target for treatment of breast and prostate cancers that proliferate in response to estrogens and androgens. Despite the crystallographic structures available for CYP17A1, no membrane-bound structural features of this enzyme at atomic level are available. Accumulating evidence has indicated that the interactions between bounded CYPs and membrane could contribute to the recruitment of lipophilic substrates. To this end, we have investigated the effects on structural characteristics in the presence of the membrane for CYP17A1. The MD simulation results demonstrate a spontaneous insertion process of the enzyme to the lipid. Two predominant modes of CYP17A1 in the membrane are captured, characterized by the depths of insertion and orientations of the enzyme to the membrane surface. The measured heme tilt angles show good consistence with experimental data, thereby verifying the validity of the structural models. Moreover, conformational changes induced by the membrane might have impact on the accessibility of the active site to lipophilic substrates. The dynamics of internal aromatic gate formed by Trp220 and Phe224 are suggested to regulate tunnel opening motions. The knowledge of the membrane binding characteristics could guide future experimental and computational works on membrane-bound CYPs so that various investigations of CYPs in their natural, lipid environment rather than in artificially solubilized forms may be achieved. Copyright © 2015. Published by Elsevier B.V.

Immobilization of β-galactosidase from Kluyveromyces lactis onto polymeric membrane surfaces: effect of surface characteristics.

PubMed

Güleç, Hacı Ali

2013-04-01

The aim of this study was to investigate the effects of surface characteristics of plain and plasma modified cellulose acetate (CA) membranes on the immobilization yield of β-galactosidases from Kluyveromyces lactis (KLG) and its galacto-oligosaccharide (GOS) yield, respectively. Low pressure plasma treatments involving oxygen plasma activation, plasma polymerization (PlsP) of ethylenediamine (EDA) and PlsP of 2-mercaptoethanol were used to modify plain CA membrane surfaces. KLG enzyme was immobilized onto plain and oxygen plasma treated membrane surfaces by simple adsorption. Oxygen plasma activation increased the hydrophylicity of CA membrane surfaces and it improved the immobilization yield of the enzyme by 42%. KLG enzyme was also immobilized onto CA membrane surfaces through amino groups created by PlsP of EDA via covalent binding. Plasma action at 60W plasma power and 15 min. exposure time improved the amount of membrane bounded enzyme by 3.5-fold. The enrichment of the amount of amino groups via polyethyleneimine (PEI) addition enhanced this increase from 3.5-fold to 4.5-fold. Although high enzyme loading was achived (65-83%), both of the methods dramatically decreased the enzyme activity (11-12%) and GOS yield due to probably negative effects of active amino groups. KLG enzyme was more effectively immobilized onto thiolated CA membrane surface created by PlsP of 2-mercaptoethanol with high immobilization yield (70%) and especially high enzyme activity (46%). Immobilized enzymes on the CA membranes treated by PlsP were successively reutilized for 5-8 cycles at 25°C and enzymatic derivatives retained approximately 75-80% of their initial activites at the end of the reactions. Copyright © 2012 Elsevier B.V. All rights reserved.

TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation.

PubMed

Reis, Carlos R; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L

2017-01-17

Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand-death receptor (TRAIL-DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL-DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL-DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors.

TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation

PubMed Central

Reis, Carlos R.; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L.

2017-01-01

Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand–death receptor (TRAIL–DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL–DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL–DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors. PMID:28049841

40 CFR 265.1086 - Standards: Surface impoundments.

Code of Federal Regulations, 2010 CFR

2010-07-01

... floating membrane cover in accordance with the provisions specified in paragraph (c) of this section; or (2... emissions from a surface impoundment using a floating membrane cover shall meet the requirements specified... with a floating membrane cover designed to meet the following specifications: (i) The floating membrane...

Dishevelled-induced phosphorylation regulates membrane localization of Par1b

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terabayashi, Takeshi; Funato, Yosuke; Miki, Hiroaki, E-mail: hmiki@protein.osaka-u.ac.jp

2008-10-31

Par1b is an evolutionarily conserved kinase that plays crucial roles in cell polarity. Controlling intracellular localization of Par1b is important for its biological activity. We previously reported that Wnt stimulation or expression of Dvl promotes accumulation of Par1b in the membrane (T. Terabayashi, T.J. Itoh, H. Yamaguchi, Y. Yoshimura, Y. Funato, S. Ohno, H. Miki, Polarity-Regulating Kinase Partitioning-Defective 1/Microtubule Affinity-Regulating Kinase 2 Negatively Regulates Development of Dendrites on Hippocampal Neurons, J. Neurosci. 27 (2007) 13098-13107). However, its molecular mechanism remains unclear. Here we show the importance of Par1b phosphorylation in the regulation of membrane localization. We find that Thr-324 ismore » phosphorylated in a Dvl-dependent manner. Interestingly, the conversion of Thr-324 to Glu results in a significant accumulation of Par1b in the membrane, without any effects on the kinase activity. Moreover, the phospho-mimicking Par1b mutant does not antagonistically function against Dvl in microtubule stabilization and neurite extension, although wildtype Par1b does. These results suggest that membrane accumulation of Par1b induced by Dvl is regulated by its phosphorylation status, which is important for Par1b to regulate the microtubule dynamics.« less

Endoplasmic Reticulum-Plasma Membrane Contacts Regulate Cellular Excitability.

PubMed

Dickson, Eamonn J

2017-01-01

Cells that have intrinsic electrical excitability utilize changes in membrane potential to communicate with neighboring cells and initiate cellular cascades. Excitable cells like neurons and myocytes have evolved highly specialized subcellular architectures to translate these electrical signals into cellular events. One such structural specialization is sarco-/endoplasmic reticulum-plasma membrane contact sites. These membrane contact sites are positioned by specific membrane-membrane tethering proteins and contain an ever-expanding list of additional proteins that organize information transfer across the junctional space (~ 15-25 nm distance) to shape membrane identity and control cellular excitability. In this chapter we discuss how contacts between the sarco-/endoplasmic reticulum and plasma membrane are essential for regulated excitation-contraction coupling in striated muscle and control of lipid-dependent ion channels.

Microbial Attachment Inhibition through Low-Voltage Electrochemical Reactions on Electrically Conducting Membranes.

PubMed

Ronen, Avner; Duan, Wenyan; Wheeldon, Ian; Walker, Sharon; Jassby, David

2015-11-03

Bacterial biofilm formation on membrane surfaces remains a serious challenge in water treatment systems. The impact of low voltages on microbial attachment to electrically conducting ultrafiltration membranes was investigated using a direct observation cross-flow membrane system mounted on a fluorescence microscope. Escherichia coli and microparticle deposition and detachment rates were measured as a function of the applied electrical potential to the membrane surface. Selecting bacteria and particles with low surface charge minimized electrostatic interactions between the bacteria and charged membrane surface. Application of an electrical potential had a significant impact on the detachment of live bacteria in comparison to dead bacteria and particles. Image analysis indicated that when a potential of 1.5 V was applied to the membrane/counter electrode pair, the percent of dead bacteria was 32±2.1 and 67±3.6% when the membrane was used as a cathode or anode, respectively, while at a potential of 1 V, 92±2.4% were alive. The application of low electrical potentials resulted in the production of low (μM) concentrations of hydrogen peroxide (HP) through the electroreduction of oxygen. The electrochemically produced HP reduced microbial cell viability and increased cellular permeability. Exposure to low concentrations of electrochemically produced HP on the membrane surface prevents bacterial attachment, thus ensuring biofilm-free conditions during membrane filtration operations.

Antifouling Thin-Film Composite Membranes by Controlled Architecture of Zwitterionic Polymer Brush Layer.

PubMed

Liu, Caihong; Lee, Jongho; Ma, Jun; Elimelech, Menachem

2017-02-21

In this study, we demonstrate a highly antifouling thin-film composite (TFC) membrane by grafting a zwitterionic polymer brush via atom-transfer radical-polymerization (ATRP), a controlled, environmentally benign chemical process. Initiator molecules for polymerization were immobilized on the membrane surface by bioinspired catechol chemistry, leading to the grafting of a dense zwitterionic polymer brush layer. Surface characterization revealed that the modified membrane exhibits reduced surface roughness, enhanced hydrophilicity, and lower surface charge. Chemical force microscopy demonstrated that the modified membrane displayed foulant-membrane interaction forces that were 1 order of magnitude smaller than those of the pristine TFC membrane. The excellent fouling resistance imparted by the zwitterionic brush layer was further demonstrated by significantly reduced adsorption of proteins and bacteria. In addition, forward osmosis fouling experiments with a feed solution containing a mixture of organic foulants (bovine-serum albumin, alginate, and natural organic matter) indicated that the modified membrane exhibited significantly lower water flux decline compared to the pristine TFC membrane. The controlled architecture of the zwitterionic polymer brush via ATRP has the potential for a facile antifouling modification of a wide range of water treatment membranes without compromising intrinsic transport properties.

The ubiquitin–proteasome system regulates membrane fusion of yeast vacuoles

PubMed Central

Kleijnen, Maurits F; Kirkpatrick, Donald S; Gygi, Steven P

2007-01-01

Ubiquitination is known to regulate early stages of intracellular vesicular transport, without proteasomal involvement. We now show that, in yeast, ubiquitination regulates a late-stage, membrane fusion, with proteasomal involvement. A known proteasome mutant had a vacuolar fragmentation phenotype in vivo often associated with vacuolar membrane fusion defects, suggesting a proteasomal role in fusion. Inhibiting vacuolar proteasomes interfered with membrane fusion in vitro, showing that fusion cannot occur without proteasomal degradation. If so, one would expect to find ubiquitinated proteins on vacuolar membranes. We found a small number of these, identified the most prevalent one as Ypt7 and mapped its two major ubiquitination sites. Ubiquitinated Ypt7 was linked to the degradation event that is necessary for fusion: vacuolar Ypt7 and vacuolar proteasomes were interdependent, ubiquitinated Ypt7 became a proteasomal substrate during fusion, and proteasome inhibitors reduced fusion to greater degree when we decreased Ypt7 ubiquitination. The strongest model holds that fusion cannot proceed without proteasomal degradation of ubiquitinated Ypt7. As Ypt7 is one of many Rab GTPases, ubiquitin–proteasome regulation may be involved in membrane fusion elsewhere. PMID:17183369

The chemical modification and characterization of polypropylene membrane with environment response by in-situ chlorinating graft copolymerization

NASA Astrophysics Data System (ADS)

Zhang, Yue; Liu, Jiankai; Hu, Wenjie; Feng, Ying; Zhao, Jiruo

2017-08-01

In this study, a novel chemical surface modification method of polyolefin membranes is applied following the in-situ chlorinating graft copolymerization (ISCGC). Polypropylene (PP)/methyl methacrylate (MMA) system was used as an example. A unique structure was formed by the modification process on the original membrane surface and the product exhibited an environmental response. Chlorine free radicals were generated using ultraviolet and heat and were used to capture the hydrogen in the polymer chains on the substrate surface. The formed macromolecular radicals could react with MMA over 2 h to achieve a high coverage ratio polymer on the PP membrane surface. The graft copolymers were characterized using FTIR, 1H-NMR, DSC, and XPS, which all proved the feasibility of chemically modifying the PP membrane surface by ISCGC. The surface morphology of the grafted PP membrane was characterized using SEM and AFM. The results showed that the grafted product presents a uniform, neat, and dense mastoid structure with an average thickness of 4.44 μm, which was expected to be similar to the brush-like surface structure. The contact angle and AFM tests indicated that the product surface is responsive to solvent and pH. The experimental results showed that the PP membrane surface structure can be reconstructed using ISCGC, a method that can be used for environment-responsive polymer materials. Moreover, the product has the characteristics of polymer interfacial brush.

S-acylation dependent post-translational cross-talk regulates large conductance calcium- and voltage- activated potassium (BK) channels

PubMed Central

Shipston, Michael J.

2014-01-01

Mechanisms that control surface expression and/or activity of large conductance calcium-activated potassium (BK) channels are important determinants of their (patho)physiological function. Indeed, BK channel dysfunction is associated with major human disorders ranging from epilepsy to hypertension and obesity. S-acylation (S-palmitoylation) represents a major reversible, post-translational modification controlling the properties and function of many proteins including ion channels. Recent evidence reveals that both pore-forming and regulatory subunits of BK channels are S-acylated and control channel trafficking and regulation by AGC-family protein kinases. The pore-forming α-subunit is S-acylated at two distinct sites within the N- and C-terminus, each site being regulated by different palmitoyl acyl transferases (zDHHCs) and acyl thioesterases (APTs). S-acylation of the N-terminus controls channel trafficking and surface expression whereas S-acylation of the C-terminal domain determines regulation of channel activity by AGC-family protein kinases. S-acylation of the regulatory β4-subunit controls ER exit and surface expression of BK channels but does not affect ion channel kinetics at the plasma membrane. Furthermore, a significant number of previously identified BK-channel interacting proteins have been shown, or are predicted to be, S-acylated. Thus, the BK channel multi-molecular signaling complex may be dynamically regulated by this fundamental post-translational modification and thus S-acylation likely represents an important determinant of BK channel physiology in health and disease. PMID:25140154

Receptor-like kinases as surface regulators for RAC/ROP-mediated pollen tube growth and interaction with the pistil

PubMed Central

Zou, Yanjiao; Aggarwal, Mini; Zheng, Wen-Guang; Wu, Hen-Ming; Cheung, Alice Y.

2011-01-01

Background RAC/ROPs are RHO-type GTPases and are known to play diverse signalling roles in plants. Cytoplasmic RAC/ROPs are recruited to the cell membrane and activated in response to extracellular signals perceived and mediated by cell surface-located signalling assemblies, transducing the signals to regulate cellular processes. More than any other cell types in plants, pollen tubes depend on continuous interactions with an extracellular environment produced by their surrounding tissues as they grow within the female organ pistil to deliver sperm to the female gametophyte for fertilization. Scope We review studies on pollen tube growth that provide compelling evidence indicating that RAC/ROPs are crucial for regulating the cellular processes that underlie the polarized cell growth process. Efforts to identify cell surface regulators that mediate extracellular signals also point to RAC/ROPs being the molecular switches targeted by growth-regulating female factors for modulation to mediate pollination and fertilization. We discuss a large volume of work spanning more than two decades on a family of pollen-specific receptor kinases and some recent studies on members of the FERONIA family of receptor-like kinases (RLKs). Significance The research described shows the crucial roles that two RLK families play in transducing signals from growth regulatory factors to the RAC/ROP switch at the pollen tube apex to mediate and target pollen tube growth to the female gametophyte and signal its disintegration to achieve fertilization once inside the female chamber. PMID:22476487

ESTIMATION OF EFFECTIVE SHEAR STRESS WORKING ON FLAT SHEET MEMBRANE USING FLUIDIZED MEDIA IN MBRs

NASA Astrophysics Data System (ADS)

Zaw, Hlwan Moe; Li, Tairi; Nagaoka, Hiroshi; Mishima, Iori

This study was aimed at estimating effective shear stress working on flat sheet membrane by the addition of fluidized media in MBRs. In both of laboratory-scale aeration tanks with and without fluidized media, shear stress variations on membrane surface and water phase velocity variations were measured and MBR operation was conducted. For the evaluation of the effective shear stress working on membrane surface to mitigate membrane surface, simulation of trans-membrane pressure increase was conducted. It was shown that the time-averaged absolute value of shear stress was smaller in the reactor with fluidized media than without fluidized media. However, due to strong turbulence in the reactor with fluidized media caused by interaction between water-phase and media and also due to the direct interaction between membrane surface and fluidized media, standard deviation of shear stress on membrane surface was larger in the reactor with fluidized media than without media. Histograms of shear stress variation data were fitted well to normal distribution curves and mean plus three times of standard deviation was defined to be a maximum shear stress value. By applying the defined maximum shear stress to a membrane fouling model, trans-membrane pressure curve in the MBR experiment was simulated well by the fouling model indicting that the maximum shear stress, not time-averaged shear stress, can be regarded as an effective shear stress to prevent membrane fouling in submerged flat-sheet MBRs.

Activation of Basophils Is a New and Sensitive Marker of Biocompatibility in Hemodialysis

PubMed Central

Aljadi, Zenib; Mansouri, Ladan; Nopp, Anna; Paulsson, Josefin M; Winqvist, Ola; Russom, Aman; Ståhl, Mårten; Hylander, Britta; Jacobson, Stefan H; Lundahl, Joachim

2014-01-01

The hemodialysis procedure involves contact between peripheral blood and the surface of dialyzer membranes, which may lead to alterations in the pathways of innate and adaptive immunity. We aimed to study the effect of blood–membrane interaction on human peripheral basophils and neutrophils in hemodialysis with high- and low-permeability polysulfone dialyzers. The surface expression of CD203c (basophil selection marker) and CD63 (activation marker) after activation by the bacterial peptide formyl-methionyl-leucyl-phenylalanine (fMLP) or anti-Fcε receptor I (FcεRI) antibody and the absolute number of basophils was investigated before and after hemodialysis with each of the dialyzers. Moreover, the expression on neutrophils of CD11b, the CD11b active epitope, and CD88 was analyzed in the same groups of individuals. The expression of CD63 in basophils following activation by fMLP was significantly higher in the patient group compared with that in healthy controls, but no differences were observed after activation by anti-FcεRI. During the hemodialysis procedure, the low-flux membrane induced up-regulation of CD63 expression on basophils, while passage through the high-flux membrane did not significantly alter the responsiveness. In addition, the absolute number of basophils was unchanged after hemodialysis with either of the dialyzers and compared with healthy controls. We found no significant differences in the expression of the neutrophil activation markers (CD11b, the active epitope of CD11b, and CD88) comparing the two different dialyzers before and after dialysis and healthy controls. Together, these findings suggest that alterations in basophil activity may be a useful marker of membrane bioincompatibility in hemodialysis. PMID:24712758

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

Functional TASK-3-Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells.

PubMed

Yao, Junlan; McHedlishvili, David; McIntire, William E; Guagliardo, Nick A; Erisir, Alev; Coburn, Craig A; Santarelli, Vincent P; Bayliss, Douglas A; Barrett, Paula Q

2017-08-01

Ca 2+ drives aldosterone synthesis in the cytosolic and mitochondrial compartments of the adrenal zona glomerulosa cell. Membrane potential across each of these compartments regulates the amplitude of the Ca 2+ signal; yet, only plasma membrane ion channels and their role in regulating cell membrane potential have garnered investigative attention as pathological causes of human hyperaldosteronism. Previously, we reported that genetic deletion of TASK-3 channels (tandem pore domain acid-sensitive K + channels) from mice produces aldosterone excess in the absence of a change in the cell membrane potential of zona glomerulosa cells. Here, we report using yeast 2-hybrid, immunoprecipitation, and electron microscopic analyses that TASK-3 channels are resident in mitochondria, where they regulate mitochondrial morphology, mitochondrial membrane potential, and aldosterone production. This study provides proof of principle that mitochondrial K + channels, by modulating inner mitochondrial membrane morphology and mitochondrial membrane potential, have the ability to play a pathological role in aldosterone dysregulation in steroidogenic cells. © 2017 American Heart Association, Inc.

Constructing dual-defense mechanisms on membrane surfaces by synergy of PFSA and SiO2 nanoparticles for persistent antifouling performance

NASA Astrophysics Data System (ADS)

Zhou, Linjie; Gao, Kang; Jiao, Zhiwei; Wu, Mengyuan; He, Mingrui; Su, Yanlei; Jiang, Zhongyi

2018-05-01

Synthetic antifouling membrane surfaces with dual-defense mechanisms (fouling-resistant and fouling-release mechanism) were constructed through the synergy of perfluorosulfonic acid (PFSA) and SiO2 nanoparticles. During the nonsolvent induced phase separation (NIPS) process, the amphiphilic PFSA polymers spontaneously segregated to membrane surfaces and catalyzed the hydrolysis-polycondensation of tetraethyl orthosilicate (TEOS) to generate hydrophilic SiO2 nanoparticles (NPs). The resulting PVDF/PFSA/SiO2 hybrid membranes were characterized by contact angle measurements, FTIR, XPS, SEM, AFM, TGA, and TEM. The hydrophilic microdomains and low surface energy microdomains of amphiphilic PFSA polymers respectively endowed membrane surfaces with fouling-resistant mechanism and fouling-release mechanism, while the hydrophilic SiO2 NPs intensified the fouling-resistant mechanism. When the addition of TEOS reached 3 wt%, the hybrid membrane with optimal synergy of PFSA and SiO2 NPs displayed low flux decline (17.4% DRt) and high flux recovery (99.8% FRR) during the filtration of oil-in-water emulsion. Meanwhile, the long-time stability test verified that the hybrid membrane possessed persistent antifouling performance.

Interfacial preparation and optical transmission surface plasmon resonance of Janus metamaterials membrane

NASA Astrophysics Data System (ADS)

Du, Yixuan; Zhang, Xiaowei; Li, Yunbo

2018-01-01

Janus metamaterials membrane had been fabricated using self-assembly strategy at the oil/water interface with thiol-terminated polymers. Janus metamaterials membrane exhibits a characteristic surface plasmon absorption band, in which the peak position is sensitive to the addition of polymer. The optical transmission surface plasmon resonance (T-SPR) peak has a blue shift at the visible region with addition of thiol-terminated polystyrene (PS-SH). With thiol-terminated poly (ethylene glycol) (PEG-SH) attachment onto the surface side of gold nanoparticles (AuNPs), the T-SPR band has a successive blue shift. One surprising thing is that it has a flat terrace on T-SPR band from 580 to 740 nm. In addition, The T-SPR of Janus metamaterials membrane dramatically changed with the addition PS-SH when the PEG-SH was capped on the opposite side. The morphologies of AuNPs membrane and Janus metamaterials membrane support the above mentioned result of SPR. In virtue of tunable SPR band, the Janus metamaterials membrane has great potential application in science-based design of optical sensing sensors and surface-enhanced optic sensitive detection.

Lytic agents, cell permeability, and monolayer penetrability.

PubMed

Salton, M R

1968-07-01

Cell lysis induced by lytic agents is the terminal phase of a series of events leading to membrane disorganization and breadkdown with the release of cellular macromolecules. Permeability changes following exposure to lytic systems may range from selective effects on ion fluxes to gross membrane damage and cell leakage. Lysis can be conceived as an interfacial phenomenon, and the action of surface-active agents on erythrocytes has provided a model in which to investigate relationships between hemolysis and chemical structure, ionic charge, surface tension lowering, and ability to penetrate monolayers of membrane lipid components. Evidence suggests that lysis follows the attainment of surface pressures exceeding a "critical collapse" level and could involve membrane cholesterol or phospholipid. Similarities of chemical composition of membranes from various cell types could account for lytic responses observed on interaction with surface-active agents. Cell membranes usually contain about 20-30 % lipid and 50-75 % protein. One or two major phospholipids are present in all cell membranes, but sterols are not detectable in bacterial membranes other than those of the Mycoplasma group. The rigid cell wall in bacteria has an important bearing on their response to treatment with lytic agents. Removal of the wall renders the protoplast membrane sensitive to rapid lysis with surfactants. Isolated membranes of erythrocytes and bacteria are rapidly dissociated by surface-active agents. Products of dissociation of bacterial membranes have uniform behavior in the ultracentrifuge (sedimentation coefficients 2-3S). Dissociation of membrane proteins from lipids and the isolation and characterization of these proteins will provide a basis for investigating the specificity of interaction of lytic agents with biomembranes.

Intravacuolar Membranes Regulate CD8 T Cell Recognition of Membrane-Bound Toxoplasma gondii Protective Antigen.

PubMed

Lopez, Jodie; Bittame, Amina; Massera, Céline; Vasseur, Virginie; Effantin, Grégory; Valat, Anne; Buaillon, Célia; Allart, Sophie; Fox, Barbara A; Rommereim, Leah M; Bzik, David J; Schoehn, Guy; Weissenhorn, Winfried; Dubremetz, Jean-François; Gagnon, Jean; Mercier, Corinne; Cesbron-Delauw, Marie-France; Blanchard, Nicolas

2015-12-15

Apicomplexa parasites such as Toxoplasma gondii target effectors to and across the boundary of their parasitophorous vacuole (PV), resulting in host cell subversion and potential presentation by MHC class I molecules for CD8 T cell recognition. The host-parasite interface comprises the PV limiting membrane and a highly curved, membranous intravacuolar network (IVN) of uncertain function. Here, using a cell-free minimal system, we dissect how membrane tubules are shaped by the parasite effectors GRA2 and GRA6. We show that membrane association regulates access of the GRA6 protective antigen to the MHC I pathway in infected cells. Although insertion of GRA6 in the PV membrane is key for immunogenicity, association of GRA6 with the IVN limits presentation and curtails GRA6-specific CD8 responses in mice. Thus, membrane deformations of the PV regulate access of antigens to the MHC class I pathway, and the IVN may play a role in immune modulation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Brownian dynamics simulations of simplified cytochrome c molecules in the presence of a charged surface

NASA Astrophysics Data System (ADS)

Gorba, C.; Geyer, T.; Helms, V.

2004-07-01

Simulations were performed for up to 150 simplified spherical horse heart cytochrome c molecules in the presence of a charged surface, which serves as an approximate model for a lipid membrane. Screened electrostatic and short-ranged attractive as well as repulsive van der Waals forces for interparticle and particle-membrane interactions are utilized in the simulations. At a distance from the membrane, where particle-membrane interactions are negligible, the simulation is coupled to a noninteraction continuum analogous to a heat bath [Geyer et al., J. Chem. Phys. 120, 4573 (2004)]. From the particles' density profiles perpendicular to the planar surface binding isotherms are derived and compared to experimental results [Heimburg et al. (1999)]. Using a negatively charged structureless membrane surface a saturation effect was found for relatively large particle concentrations. Since biological membranes often contain membrane proteins, we also studied the influence of additional charges on our model membrane mimicking bacterial reaction centers. We find that the onset of the saturation occurs for much lower concentrations and is sensitive to the detailed implementation. Therefore we suggest that local distortion of membrane planarity (undulation), or lipid demixing, or the presence of charged integral membrane proteins create preferential binding sites on the membrane. Only then do we observe saturation at physiological concentrations.

SECRETED KLOTHO AND CHRONIC KIDNEY DISEASE

PubMed Central

Hu, Ming Chang; Kuro-o, Makoto; Moe, Orson W.

2013-01-01

Soluble Klotho (sKl) in the circulation can be generated directly by alterative splicing of the Klotho transcript or the extracellular domain of membrane Klotho can be released from membrane-anchored Klotho on the cell surface. Unlike membrane Klotho which functions as a coreceptor for fibroblast growth factor-23 (FGF23), sKl, acts as hormonal factor and plays important roles in anti-aging, anti-oxidation, modulation of ion transport, and Wnt signaling. Emerging evidence reveals that Klotho deficiency is an early biomarker for chronic kidney diseases as well as a pathogenic factor. Klotho deficiency is associated with progression and chronic complications in chronic kidney disease including vascular calcification, cardiac hypertrophy, and secondary hyperparathyroidism. In multiple experimental models, replacement of sKl, or manipulated up-regulation of endogenous Klotho protect the kidney from renal insults, preserve kidney function, and suppress renal fibrosis, in chronic kidney disease. Klotho is a highly promising candidate on the horizon as an early biomarker, and as a novel therapeutic agent for chronic kidney disease. PMID:22396167

Membrane Cholesterol Modulates Superwarfarin Toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marangoni, M. Natalia; Martynowycz, Michael W.; Kuzmenko, Ivan

Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but notmore » warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.« less

Tuning B cell responses to antigens by cell polarity and membrane trafficking.

PubMed

Del Valle Batalla, Felipe; Lennon-Dumenil, Ana-María; Yuseff, María-Isabel

2018-06-20

The capacity of B lymphocytes to produce specific antibodies, particularly broadly neutralizing antibodies that provide immunity to viral pathogens has positioned them as valuable therapeutic targets for immunomodulation. To become competent as antibody secreting cells, B cells undergo a series of activation steps, which are triggered by the recognition of antigens frequently displayed on the surface of other presenting cells. Such antigens elicit the formation of an immune synapse (IS), where local cytoskeleton rearrangements coupled to mechanical forces and membrane trafficking orchestrate the extraction and processing of antigens in B cells. In this review, we discuss the molecular mechanisms that regulate polarized membrane trafficking and mechanical properties of the immune synapse, as well as the potential extracellular cues from the environment, which may impact the ability of B cells to sense and acquire antigens at the immune synapse. An integrated view of the diverse cellular mechanisms that shape the immune synapse will provide a better understanding on how B cells are efficiently activated. Copyright © 2018 Elsevier Ltd. All rights reserved.

Plant nutrition: root transporters on the move.

PubMed

Zelazny, Enric; Vert, Grégory

2014-10-01

Nutrient and water uptake from the soil is essential for plant growth and development. In the root, absorption and radial transport of nutrients and water toward the vascular tissues is achieved by a battery of specialized transporters and channels. Modulating the amount and the localization of these membrane transport proteins appears as a way to drive their activity and is essential to maintain nutrient homeostasis in plants. This control first involves the delivery of newly synthesized proteins to the plasma membrane by establishing check points along the secretory pathway, especially during the export from the endoplasmic reticulum. Plasma membrane-localized transport proteins are internalized through endocytosis followed by recycling to the cell surface or targeting to the vacuole for degradation, hence constituting another layer of control. These intricate mechanisms are often regulated by nutrient availability, stresses, and endogenous cues, allowing plants to rapidly adjust to their environment and adapt their development. © 2014 American Society of Plant Biologists. All Rights Reserved.

Switchable hydrophobic/hydrophilic surface of electrospun poly (l-lactide) membranes obtained by CF₄microwave plasma treatment

DOE PAGES

Yue, Mengyao; Zhou, Baoming; Jiao, Kunyan; ...

2014-11-29

A switchable surface that promotes either hydrophobic or hydrophilic wettability of poly (L-lactide) (PLLA) microfibrous membranes is obtained by CF₄ microwave plasma treatment in this paper. The results indicated that both etching and grafting process occurred during the CF₄ plasma treatment and these two factors synergistically affected the final surface wettability of PLLA membranes. When plasma treatment was taken under a relatively low power, the surface wettability of PLLA membranes turned from hydrophobic to hydrophilic. Especially when CF₄ plasma treatment was taken under 100 W for 10 min and 150 W for 5 min, the water contact angle sharply decreasedmore » from 116 ± 3.0° to ~0°. According to Field-emission scanning electron microscopy (FESEM) results, the PLLA fibers were notably etched by CF₄ plasma treatment. Combined with the X-ray photoelectron spectroscopy (XPS) measurements, only a few fluorine-containing groups were grafted onto the surface, so the etching effect directly affected the surface wettability of PLLA membranes in low plasma power condition. However, with the plasma power increasing to 200 W, the PLLA membrane surface turned to hydrophobic again. In contrast, the morphology changes of PLLA fiber surfaces were not obvious while a large number of fluorine-containing groups grafted onto the surface. So the grafting effect gradually became the major factor for the final surface wettability.« less

ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity.

PubMed

Daley, William P; Gervais, Elise M; Centanni, Samuel W; Gulfo, Kathryn M; Nelson, Deirdre A; Larsen, Melinda

2012-01-01

The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.

CuSO4/H2O2-Triggered Polydopamine/Poly(sulfobetaine methacrylate) Coatings for Antifouling Membrane Surfaces.

PubMed

Zhang, Chao; Li, Hao-Nan; Du, Yong; Ma, Meng-Qi; Xu, Zhi-Kang

2017-02-07

Mussel-inspired polydopamine (PDA) coatings have been broadly exploited for constructing functional membrane surfaces. One-step codeposition of PDA with antifouling polymers, especially zwitterionic polymers, has been regarded as a promising strategy for fabricating antifouling membrane surfaces. However, one challenge is that the codeposition is usually a slow process over 10 h or even several days. Herein, we report that CuSO 4 /H 2 O 2 is able to notably accelerate the codeposition process of PDA with poly(sulfobetaine methacrylate) (PSBMA). In our case, PSBMA is facilely anchored to the polypropylene microporous membrane (PPMM) surfaces within 1 h with the assistance of PDA because of its strong interfacial adhesion. The PDA/PSBMA-coated PPMMs show excellent surface hydrophilicity, high water permeation flux (7506 ± 528 L/m 2 ·h at 0.1 MPa), and an outstanding antifouling property. Moreover, the antifouling property is maintained after the membranes are treated with acid and alkali solutions as well as organic solvents. To recap, it provides a facile, universal, and time-saving strategy for exploiting high-efficiency and durable antifouling membrane surfaces.

Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction.

PubMed

Heuser, J E; Reese, T S

1973-05-01

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae.

EVIDENCE FOR RECYCLING OF SYNAPTIC VESICLE MEMBRANE DURING TRANSMITTER RELEASE AT THE FROG NEUROMUSCULAR JUNCTION

PubMed Central

Heuser, J. E.; Reese, T. S.

1973-01-01

When the nerves of isolated frog sartorius muscles were stimulated at 10 Hz, synaptic vesicles in the motor nerve terminals became transiently depleted. This depletion apparently resulted from a redistribution rather than disappearance of synaptic vesicle membrane, since the total amount of membrane comprising these nerve terminals remained constant during stimulation. At 1 min of stimulation, the 30% depletion in synaptic vesicle membrane was nearly balanced by an increase in plasma membrane, suggesting that vesicle membrane rapidly moved to the surface as it might if vesicles released their content of transmitter by exocytosis. After 15 min of stimulation, the 60% depletion of synaptic vesicle membrane was largely balanced by the appearance of numerous irregular membrane-walled cisternae inside the terminals, suggesting that vesicle membrane was retrieved from the surface as cisternae. When muscles were rested after 15 min of stimulation, cisternae disappeared and synaptic vesicles reappeared, suggesting that cisternae divided to form new synaptic vesicles so that the original vesicle membrane was now recycled into new synaptic vesicles. When muscles were soaked in horseradish peroxidase (HRP), this tracerfirst entered the cisternae which formed during stimulation and then entered a large proportion of the synaptic vesicles which reappeared during rest, strengthening the idea that synaptic vesicle membrane added to the surface was retrieved as cisternae which subsequently divided to form new vesicles. When muscles containing HRP in synaptic vesicles were washed to remove extracellular HRP and restimulated, HRP disappeared from vesicles without appearing in the new cisternae formed during the second stimulation, confirming that a one-way recycling of synaptic membrane, from the surface through cisternae to new vesicles, was occurring. Coated vesicles apparently represented the actual mechanism for retrieval of synaptic vesicle membrane from the plasma membrane, because during nerve stimulation they proliferated at regions of the nerve terminals covered by Schwann processes, took up peroxidase, and appeared in various stages of coalescence with cisternae. In contrast, synaptic vesicles did not appear to return directly from the surface to form cisternae, and cisternae themselves never appeared directly connected to the surface. Thus, during stimulation the intracellular compartments of this synapse change shape and take up extracellular protein in a manner which indicates that synaptic vesicle membrane added to the surface during exocytosis is retrieved by coated vesicles and recycled into new synaptic vesicles by way of intermediate cisternae. PMID:4348786

A novel membrane anchor for FtsZ is linked to cell wall hydrolysis in Caulobacter crescentus.

PubMed

Meier, Elizabeth L; Razavi, Shiva; Inoue, Takanari; Goley, Erin D

2016-07-01

In most bacteria, the tubulin-like GTPase FtsZ forms an annulus at midcell (the Z-ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z-ring assembly and early FtsZ-directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C-terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane-anchored FtsZ in the regulation of cell wall hydrolysis. © 2016 John Wiley & Sons Ltd.

Carbon nanotube embedded PVDF membranes: Effect of solvent composition on the structural morphology for membrane distillation

NASA Astrophysics Data System (ADS)

Mapunda, Edgar C.; Mamba, Bhekie B.; Msagati, Titus A. M.

2017-08-01

Rapid population increase, growth in industrial and agricultural sectors and global climate change have added significant pressure on conventional freshwater resources. Tapping freshwater from non-conventional water sources such as desalination and wastewater recycling is considered as sustainable alternative to the fundamental challenges of water scarcity. However, affordable and sustainable technologies need to be applied for the communities to benefit from the treatment of non-conventional water source. Membrane distillation is a potential desalination technology which can be used sustainably for this purpose. In this work multi-walled carbon nanotube embedded polyvinylidene fluoride membranes for application in membrane distillation desalination were prepared via non-solvent induced phase separation method. The casting solution was prepared using mixed solvents (N, N-dimethylacetamide and triethyl phosphate) at varying ratios to study the effect of solvent composition on membrane morphological structures. Membrane morphological features were studied using a number of techniques including scanning electron microscope, atomic force microscope, SAXSpace tensile strength analysis, membrane thickness, porosity and contact angle measurements. It was revealed that membrane hydrophobicity, thickness, tensile strength and surface roughness were increasing as the composition of N, N-dimethylacetamide in the solvent was increasing with maximum values obtained between 40 and 60% N, N-dimethylacetamide. Internal morphological structures were changing from cellular structures to short finger-like and sponge-like pores and finally to large macro void type of pores when the amount of N, N-dimethylacetamide in the solvent was changed from low to high respectively. Multi-walled carbon nanotube embedded polyvinylidene fluoride membranes of desired morphological structures and physical properties can be synthesized by regulating the composition of solvents used to prepare the casting solution.

Fouling potential evaluation of soluble microbial products (SMP) with different membrane surfaces in a hybrid membrane bioreactor using worm reactor for sludge reduction.

PubMed

Li, Zhipeng; Tian, Yu; Ding, Yi; Chen, Lin; Wang, Haoyu

2013-07-01

The fouling characteristics of soluble microbial products (SMP) in the membrane bioreactor coupled with Static Sequencing Batch Worm Reactor (SSBWR-MBR) were tested with different types of membranes. It was noted that the flux decrements of S-SMP (SMP in SSBWR-MBR) with cellulose acetate (CA), polyvinylidene fluoride (PVDF) and polyether sulfones (PES) membranes were respectively 6.7%, 8.5% and 9.5% lower compared to those of C-SMP (SMP in Control-MBR) with corresponding membranes. However, for both the filtration of the C-SMP and S-SMP, the CA membrane exhibited the fastest diminishing rate of flux among the three types of membranes. The surface morphology analysis showed that the CA membrane exhibited more but smaller protuberances compared to the PVDF and PES. The second minimums surrounding each protruding asperity on CA membrane were more than those on the PVDF and PES membranes, enhancing the attachment of SMP onto the membrane surface. Copyright © 2013 Elsevier Ltd. All rights reserved.

High performance membrane-electrode assembly based on a surface-modified membrane

NASA Astrophysics Data System (ADS)

Han, Sangil; Lee, Jang Woo; Kwak, Chan; Chai, Geun Seok; Son, In Hyuk; Jang, Moon Yup; An, Sung Guk; Cho, Sung Yong; Kim, Jun Young; Kim, Hyung Wook; Serov, Alexey Alexandrovych; Yoo, Youngtai; Nam, Kie Hyun

A surface-modified membrane is prepared using a sputtering technique that deposits gold directly on a Nafion ® 115 membrane surface that is roughened with silicon carbide paper. The surface-modified membranes are characterized by means of a scanning electron microscope (SEM), differential scanning calorimetry (DSC), and water contact-angle analysis. A single direct methanol fuel cell (DMFC) with a surface-modified membrane exhibits enhanced performance (160 mW cm -2), while a bare Nafion ® 115 cell yields 113 mW cm -2 at 0.4 V and an operating temperature of 70 °C. From FE-SEM images and CO ad stripping voltammograms, it is also found that the gold layer is composed of clusters of porous nodule-like particles, which indicates that an anode with nodule-like gold leads to the preferential oxidation of carbon monoxide. These results suggest that the topology of gold in the interfacial area and its electrocatalytic nature may be the critical factors that affect DMFC performance.

In Vitro Reconstitution of Microtubule Plus End-directed, GTPγS-sensitive Motility of Golgi MembranesV⃞

PubMed Central

Fullerton, Aaron T.; Bau, Mu-Yeh; Conrad, Patricia A.; Bloom, George S.

1998-01-01

Purified Golgi membranes were mixed with cytosol and microtubules (MTs) and observed by video enhanced light microscopy. Initially, the membranes appeared as vesicles that moved along MTs. As time progressed, vesicles formed aggregates from which membrane tubules emerged, traveled along MTs, and eventually generated extensive reticular networks. Membrane motility required ATP, occurred mainly toward MT plus ends, and was inhibited almost completely by the H1 monoclonal antibody to kinesin heavy chain, 5′-adenylylimidodiphosphate, and 100 μM but not 20 μM vanadate. Motility was also blocked by GTPγS or AlF4− but was insensitive to AlCl3, NaF, staurosporin, or okadaic acid. The targets for GTPγS and AlF4− were evidently of cytosolic origin, did not include kinesin or MTs, and were insensitive to several probes for trimeric G proteins. Transport of Golgi membranes along MTs mediated by a kinesin has thus been reconstituted in vitro. The motility is regulated by one or more cytosolic GTPases but not by protein kinases or phosphatases that are inhibited by staurosporin or okadaic acid, respectively. The pertinent GTPases are likely to be small G proteins or possibly dynamin. The in vitro motility may correspond to Golgi-to-ER or Golgi-to-cell surface transport in vivo. PMID:9763438

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

PubMed Central

2012-01-01

Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (−) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (−) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection. PMID:22857383

Antioxidation performance of poly(vinyl alcohol) modified poly(vinylidene fluoride) membranes

NASA Astrophysics Data System (ADS)

Wang, Daohui; Li, Xianfeng; Li, Qing; Liu, Zhen; Li, Nana; Huang, Qinglin; Zhang, Yufeng; Xiao, Changfa

2018-03-01

Commercial poly(vinylidene fluoride) (PVDF) membranes were modified by dip-coating and crosslinking hydrophilic poly(vinyl alcohol) (PVA) on the membrane surface. The antioxidation performance of the modified PVDF membranes was evaluated via exposing the modified membranes to sodium hypochlorite and potassium permanganate solutions for 5-30 days, respectively. The evaluation was based on the influences of the two oxidants on the permeability, rejection, and hydrophility of the modified membranes, which were characterized by water flux, ink rejection, water contact angle, x-ray photoelectron spectroscopy (XPS), field emission scanning electron microscopy, and x-ray diffraction (XRD) measurements. The XPS and water contact angle results show that the hydrophilicity of PVDF membranes was significantly improved when PVA was crosslinked on the surface of PVDF membranes. When the modified membranes had been treated with sodium hypochlorite or potassium permanganate for 30 days, the permeability and hydrophilicity were basically maintained and the rejection was slightly decreased. XPS and XRD indicated that some of PVAs coated on the membrane surface could be oxidized and peeled.

Novel Fouling-Reducing Coatings for Ultrafiltration, Nanofiltration, and Reverse Osmosis Membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Benny Freeman

2008-08-31

Polymeric membranes could potentially be the most flexible and viable long-term strategy for treatment of produced water from oil and gas production. However, widespread use of membranes, including reverse osmosis (RO) membranes, for produced water purification is hindered due to fouling caused by the impurities present in the water. Fouling of RO membranes is likely caused by surface properties including roughness, hydrophilicity, and charge, so surface modification is the most widely considered approach to improve the fouling properties of current RO membranes. This project focuses on two main approaches to surface modification: coating and grafting. Hydrophilic coating and grafting materialsmore » based on poly(ethylene glycol) (PEG) are applied to commercial RO membranes manufactured by Dow FilmTec and GE. Crossflow filtration experiments are used to determine the fouling resistance of modified membranes, and compare their performance to that of unmodified commercial RO membranes. Grafting and coating are shown to be two alternative methods of producing modified membranes with improved fouling resistance.« less

Surface modification of cellulose acetate membrane using thermal annealing to enhance produced water treatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kusworo, T. D., E-mail: tdkusworo@che.undip.ac.id; Aryanti, N., E-mail: nita.aryanti@gmail.com; Firdaus, M. M. H.

2015-12-29

This study is performed primarily to investigate the effect of surface modification of cellulose acetate using thermal annealing on the enhancement of membrane performance for produced water treatment. In this study, Cellulose Acetate membranes were casted using dry/wet phase inversion technique. The effect of additive and post-treatment using thermal annealing on the membrane surface were examined for produced water treatment. Therma annealing was subjected to membrane surface at 60 and 70 °C for 5, 10 and 15 second, respectively. Membrane characterizations were done using membrane flux and rejection with produced water as a feed, Scanning Electron Microscopy (SEM) and Fouriermore » Transform Infra Red (FTIR) analysis. Experimental results showed that asymmetric cellulose acetate membrane can be made by dry/wet phase inversion technique. The results from the Scanning Electron Microscopy (FESEM) analysis was also confirmed that polyethylene glycol as additivie in dope solution and thermal annealing was affected the morphology and membrane performance for produced water treatment, respectively. Scanning electron microscopy micrographs showed that the selective layer and the substructure of membrane became denser and more compact after the thermal annealing processes. Therefore, membrane rejection was significantly increased while the flux was slighty decreased, respectively. The best membrane performance is obtained on the composition of 18 wt % cellulose acetate, poly ethylene glycol 5 wt% with thermal annealing at 70° C for 15 second.« less

Superhydrophobic and superhydrophilic surface-enhanced separation performance of porous inorganic membranes for biomass-to-biofuel conversion applications

DOE PAGES

Hu, Michael Z.; Engtrakul, Chaiwat; Bischoff, Brian L.; ...

2016-11-14

A new class of inorganic-based membranes, i.e., High-Performance Architectured Surface Selective (HiPAS) membranes, is introduced to provide high perm-selective flux by exploiting unique separation mechanisms induced by superhydrophobic or superhydrophilic surface interactions and confined capillary condensation in enlarged membrane pores (~8 nm). The super-hydro-tunable HiPAS membranes were originally developed for the purpose of bio-oil/biofuel processing to achieve selective separations at higher flux relative to size selective porous membranes (e.g., inorganic zeolite-based membranes) and better high-temperature tolerance than polymer membranes (>250 C) for hot vapor processing. Due to surface-enhanced separation selectivity, HiPAS membranes can thus possibly enable larger pores to facilitatemore » large-flux separations by increasing from sub-nanometer pores to mesopores (2-50 nm) for vapor phase or micron-scale pores for liquid phase separations. In this paper, we describe an innovative membrane concept and a materials synthesis strategy to fabricate HiPAS membranes, and demonstrate selective permeation in both vapor- and liquid-phase applications. High permeability and selectivity were demonstrated using surrogate mixtures, such as ethanol-water, toluene-water, and toluene-phenol-water. The overall membrane evaluation results show promise for the future processing of biomass pyrolysis and upgraded product vapors and condensed liquid bio-oil intermediates.« less

Blood-testis barrier dynamics are regulated by testosterone and cytokines via their differential effects on the kinetics of protein endocytosis and recycling in Sertoli cells

PubMed Central

Yan, Helen H. N.; Mruk, Dolores D.; Lee, Will M.; Cheng, C. Yan

2009-01-01

During spermatogenesis in the mammalian testis, preleptotene/leptotene spermatocytes differentiate from type B spermatogonia and traverse the blood-testis barrier (BTB) at stage VIII of the seminiferous epithelial cycle for further development. This timely movement of germ cells involves extensive junction restructuring at the BTB. Previous studies have shown that these events are regulated by testosterone (T) and cytokines [e.g., the transforming growth factor (TGF) -βs], which promote and disrupt the BTB assembly, respectively. However, the mechanisms underlying the “opening” of the BTB above a migrating preleptotene/leptotene spermatocyte and the “resealing” of the barrier underneath this cell remain obscure. We now report findings on a novel mechanism utilized by the testes to regulate these events. Using cell surface protein biotinylation coupled with immunoblotting and immunofluorescent microscopy, we assessed the kinetics of endocytosis and recycling of BTB-associated integral membrane proteins: occludin, JAM-A, and N-cadherin. It was shown that these proteins were continuously endocytosed and recycled back to the Sertoli cell surface via the clathrin-mediated but not the caveolin-mediated pathway. When T or TGF-β2 was added to Sertoli cell cultures with established functional BTB, both factors accelerated the kinetics of internalization of BTB proteins from the cell surface, perhaps above the migrating preleptotene spermatocyte, thereby opening the BTB. Likewise, T also enhanced the kinetics of recycling of internalized biotinylated proteins back to the cell surface, plausibly relocating these proteins beneath the migrating spermatocyte to reassemble the BTB. In contrast, TGF-β2 targeted internalized biotinylated proteins to late endosomes for degradation, destabilizing the BTB. In summary, the transient opening of the BTB that facilitates germ cell movement is mediated via the differential effects of T and cytokines on the kinetics of endocytosis and recycling of integral membrane proteins at the BTB. The net result of these interactions, in turn, determines the steady-state protein levels at the Sertoli-Sertoli cell interface at the BTB. PMID:18192323

Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human δ-opioid receptor turnover at the plasma membrane.

PubMed

Lackman, Jarkko J; Goth, Christoffer K; Halim, Adnan; Vakhrushev, Sergey Y; Clausen, Henrik; Petäjä-Repo, Ulla E

2018-01-01

G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors at the cell surface. Copyright © 2017 Elsevier Inc. All rights reserved.

Endocytosis and Trafficking of Natriuretic Peptide Receptor-A: Potential Role of Short Sequence Motifs

PubMed Central

Pandey, Kailash N.

2015-01-01

The targeted endocytosis and redistribution of transmembrane receptors among membrane-bound subcellular organelles are vital for their correct signaling and physiological functions. Membrane receptors committed for internalization and trafficking pathways are sorted into coated vesicles. Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP) bind to guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) and elicit the generation of intracellular second messenger cyclic guanosine 3',5'-monophosphate (cGMP), which lowers blood pressure and incidence of heart failure. After ligand binding, the receptor is rapidly internalized, sequestrated, and redistributed into intracellular locations. Thus, NPRA is considered a dynamic cellular macromolecule that traverses different subcellular locations through its lifetime. The utilization of pharmacologic and molecular perturbants has helped in delineating the pathways of endocytosis, trafficking, down-regulation, and degradation of membrane receptors in intact cells. This review describes the investigation of the mechanisms of internalization, trafficking, and redistribution of NPRA compared with other cell surface receptors from the plasma membrane into the cell interior. The roles of different short-signal peptide sequence motifs in the internalization and trafficking of other membrane receptors have been briefly reviewed and their potential significance in the internalization and trafficking of NPRA is discussed. PMID:26151885

Supramolecular organization of the sperm plasma membrane during maturation and capacitation.

PubMed

Jones, Roy; James, Peter S; Howes, Liz; Bruckbauer, Andreas; Klenerman, David

2007-07-01

In the present study, a variety of high resolution microscopy techniques were used to visualize the organization and motion of lipids and proteins in the sperm's plasma membrane. We have addressed questions such as the presence of diffusion barriers, confinement of molecules to specific surface domains, polarized diffusion and the role of cholesterol in regulating lipid rafts and signal transduction during capacitation. Atomic force microscopy identified a novel region (EqSS) within the equatorial segment of bovine, porcine and ovine spermatozoa that was enriched in constitutively phosphorylated proteins. The EqSS was assembled during epididymal maturation. Fluorescence imaging techniques were then used to follow molecular diffusion on the sperm head. Single lipid molecules were freely exchangeable throughout the plasma membrane and showed no evidence for confinement within domains. Large lipid aggregates, however, did not cross over the boundary between the post-acrosome and equatorial segment suggesting the presence of a molecular filter between these two domains. A small reduction in membrane cholesterol enlarges or increases lipid rafts concomitant with phosphorylation of intracellular proteins. Excessive removal of cholesterol, however, disorganizes rafts with a cessation of phosphorylation. These techniques are forcing a revision of long-held views on how lipids and proteins in sperm membranes are assembled into larger complexes that mediate recognition and fusion with the egg.

Molecular recognition in myxobacterial outer membrane exchange: functional, social and evolutionary implications.

PubMed

Wall, Daniel

2014-01-01

Through cooperative interactions, bacteria can build multicellular communities. To ensure that productive interactions occur, bacteria must recognize their neighbours and respond accordingly. Molecular recognition between cells is thus a fundamental behaviour, and in bacteria important discoveries have been made. This MicroReview focuses on a recently described recognition system in myxobacteria that is governed by a polymorphic cell surface receptor called TraA. TraA regulates outer membrane exchange (OME), whereby myxobacterial cells transiently fuse their OMs to efficiently transfer proteins and lipids between cells. Unlike other transport systems, OME is rather indiscriminate in what OM goods are transferred. In contrast, the recognition of partnering cells is discriminatory and only occurs between cells that bear identical or closely related TraA proteins. Therefore TraA functions in kin recognition and, in turn, OME helps regulate social interactions between myxobacteria. Here, I discuss and speculate on the social and evolutionary implications of OME and suggest it helps to guide their transition from free-living cells into coherent and functional populations. © 2013 John Wiley & Sons Ltd.

IKKβ and NFκB transcription govern lymphoma cell survival through AKT-induced plasma membrane trafficking of GLUT1

PubMed Central

Sommermann, Thomas; O’Neill, Kathleen; Plas, David R.; Cahir-McFarland, Ellen

2011-01-01

All cancer cells require increased nutrient uptake to support proliferation. Here we investigated the signals that govern glucose uptake in B-cell lymphomas and determined that the protein kinase IKKβ induced GLUT1 membrane trafficking in both viral and spontaneous B-cell lymphomas. IKKβ induced AKT activity, while IKKβ-driven NFκB transcription was required for GLUT1 surface localization downstream of AKT. Activated NFκB promoted AKT-mediated phosphorylation of the GLUT1 regulator, AKT Substrate 160kD (AS160), but was not required for AKT phosphorylation of the mammalian target of rapamycin (mTOR) regulator Tuberous Sclerosis 2 (TSC2). In Epstein Barr virus (EBV) transformed B-cells, NFκB inhibition repressed glucose uptake and induced caspase-independent cell death associated with autophagy. After NFκB inhibition, an alternate carbon source ameliorated both autophagy and cell death, whereas autophagy inhibitors specifically accelerated cell death. Taken together, the results suggest that NFκB signaling establishes a metabolic program supporting proliferation and apoptosis resistance by driving glucose import. PMID:21987722

Syndecan-4 Phosphorylation Is a Control Point for Integrin Recycling

PubMed Central

Morgan, Mark R.; Hamidi, Hellyeh; Bass, Mark D.; Warwood, Stacey; Ballestrem, Christoph; Humphries, Martin J.

2013-01-01

Summary Precise spatiotemporal coordination of integrin adhesion complex dynamics is essential for efficient cell migration. For cells adherent to fibronectin, differential engagement of α5β1 and αVβ3 integrins is used to elicit changes in adhesion complex stability, mechanosensation, matrix assembly, and migration, but the mechanisms responsible for receptor regulation have remained largely obscure. We identify phosphorylation of the membrane-intercalated proteoglycan syndecan-4 as an essential switch controlling integrin recycling. Src phosphorylates syndecan-4 and, by driving syntenin binding, leads to suppression of Arf6 activity and recycling of αVβ3 to the plasma membrane at the expense of α5β1. The resultant elevation in αVβ3 engagement promotes stabilization of focal adhesions. Conversely, abrogation of syndecan-4 phosphorylation drives surface expression of α5β1, destabilizes adhesion complexes, and disrupts cell migration. These data identify the dynamic spatiotemporal regulation of Src-mediated syndecan-4 phosphorylation as an essential switch controlling integrin trafficking and adhesion dynamics to promote efficient cell migration. PMID:23453597

Two barriers for sodium in vascular endothelium?

PubMed Central

Oberleithner, Hans

2012-01-01

Vascular endothelium plays a key role in blood pressure regulation. Recently, it has been shown that a 5% increase of plasma sodium concentration (sodium excess) stiffens endothelial cells by about 25%, leading to cellular dysfunction. Surface measurements demonstrated that the endothelial glycocalyx (eGC), an anionic biopolymer, deteriorates when sodium is elevated. In view of these results, a two-barrier model for sodium exiting the circulation across the endothelium is suggested. The first sodium barrier is the eGC which selectively buffers sodium ions with its negatively charged prote-oglycans.The second sodium barrier is the endothelial plasma membrane which contains sodium channels. Sodium excess, in the presence of aldosterone, leads to eGC break-down and, in parallel, to an up-regulation of plasma membrane sodium channels. The following hypothesis is postulated: Sodium excess increases vascular sodium permeability. Under such con-ditions (e.g. high-sodium diet), day-by-day ingested sodium, instead of being readily buffered by the eGC and then rapidly excreted by the kidneys, is distributed in the whole body before being finally excreted. Gradually, the sodium overload damages the organism. PMID:22471931

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts.

PubMed

Ring, Axel; Le Lay, Soazig; Pohl, Juergen; Verkade, Paul; Stremmel, Wolfgang

2006-04-01

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.

The early endosome: a busy sorting station for proteins at the crossroads

PubMed Central

Jovic, Marko; Sharma, Mahak; Rahajeng, Juliati; Caplan, Steve

2010-01-01

Summary Endocytosis marks the entry of internalized receptors into the complex network of endocytic trafficking pathways. Endocytic vesicles are rapidly targeted to a distinct membrane-bound endocytic organelle referred to as the early endosome. Despite the existence of numerous internalization routes, early endosomes (EE) serve as a focal point of the endocytic pathway. Sorting events initiated at this compartment determine the subsequent fate of internalized proteins and lipids, destining them either for recycling to the plasma membrane, degradation in lysosomes or delivery to the trans-Golgi network. Sorting of endocytic cargo to the latter compartments is accomplished through the formation of distinct microdomains within early endosomes, through the coordinate recruitment and assembly of the sorting machinery. An elaborate network of interactions between endocytic regulatory proteins ensures synchronized sorting of cargo to microdomains followed by morphological changes at the early endosomal membranes. Consequently, the cargo targeted either for recycling back to the plasma membrane, or for retrograde transport to the trans-Golgi network, localizes to newly-formed tubular membranes. With a high ratio of membrane surface to lumenal volume, these tubules effectively concentrate the recycling cargo, ensuring efficient transport out of the EE. Conversely, receptors sorted for degradation cluster at the flat clathrin lattices involved in invaginations of the limiting membrane, associating with newly formed intralumenal vesicles. In this review we will discuss the characteristics of early endosomes, their role in the regulation of endocytic transport, and their aberrant function in a variety of diseases. PMID:19924646

Membrane targeting of WAVE2 is not sufficient for WAVE2-dependent actin polymerization: a role for IRSp53 in mediating the interaction between Rac and WAVE2.

PubMed

Abou-Kheir, Wassim; Isaac, Beth; Yamaguchi, Hideki; Cox, Dianne

2008-02-01

Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous (WAVE) proteins play a major role in Rac-induced actin dynamics, but Rac does not bind directly to WAVE proteins. It has been proposed that either the insulin receptor substrate protein 53 (IRSp53) or a complex of proteins containing Abelson interactor protein 1 (Abi1) mediates the interaction of WAVE2 and Rac. Depletion of endogenous IRSp53 by RNA-mediated interference (RNAi) in a RAW/LR5 macrophage cell line resulted in a significant reduction of Rac1Q61L-induced surface ruffles and colony-stimulating factor 1 (CSF-1)-induced actin polymerization, protrusion and cell migration. However, IRSp53 was not essential for Fcgamma-R-mediated phagocytosis, formation of podosomes or for formation of Cdc42V12-induced filopodia. IRSp53 was found to be present in an immunoprecipitable complex with WAVE2 and Abi1 in a Rac1-activation-dependent manner in RAW/LR5 cells in vivo. Importantly, reduction of endogenous IRSp53 or expression of IRSp53 lacking the WAVE2-binding site (IRSp53DeltaSH3) resulted in a significant reduction in the association of Rac1 with WAVE2 and Abi1, indicating that the association of Rac1 with WAVE2 and Abi1 is IRSp53 dependent. While it has been proposed that WAVE2 activity is regulated by membrane recruitment, membrane targeting of WAVE2 in RAW/LR5 and Cos-7 cells did not induce actin polymerization or protrusion, suggesting that membrane recruitment was insufficient for regulation of WAVE2. Combined, these data suggest that IRSp53 links Rac1 to WAVE2 in vivo and its function is crucial for production of CSF-1-induced F-actin-rich protrusions and cell migration in macrophages. This study indicates that Rac1, along with IRSp53 and Abi1, is involved in a more complex and tight regulation of WAVE2 than one operating solely through membrane localization.

Membrane targeting of WAVE2 is not sufficient for WAVE2 dependent actin polymerization: a role for IRSp53 in mediating the interaction between Rac and WAVE2*

PubMed Central

Abou-Kheir, Wassim; Isaac, Beth; Yamaguchi, Hideki; Cox, Dianne

2009-01-01

Summary Wiskott-Aldrich syndrome protein (WASP)-family verprolin homologous (WAVE) proteins play a major role in Rac-induced actin dynamics, but Rac does not bind directly to WAVE proteins. It has been proposed that either the insulin receptor substrate protein 53 (IRSp53) or a complex of proteins containing Abelson interactor protein 1 (Abi1) mediate the interaction of WAVE2 and Rac. Depletion of endogenous IRSp53 by RNA-mediated interference (RNAi) in a RAW/LR5 macrophage cell line resulted in a significant reduction of Rac1Q61L-induced surface ruffles and colony stimulating factor-1 (CSF-1)-induced actin polymerization, protrusion, and cell migration. However, IRSp53 was not essential for Fcγ-R-mediated phagocytosis, formation of podosomes or for Cdc42V12-induced filopodia. IRSp53 was found to be present in an immunoprecipitatable complex with WAVE2 and Abi1 in a Rac1 activation-dependent manner in RAW/LR5 cells in vivo. Importantly, reduction of endogenous IRSp53 or expression of IRSp53 lacking the WAVE2 binding site (IRSp53ΔSH3) resulted in a significant reduction in the association of Rac1 with WAVE2 and Abi1, indicating that the association of Rac1 with WAVE2 and Abi1 is IRSp53 dependent. While it has been proposed that WAVE2 activity is regulated by membrane recruitment, membrane targeting of WAVE2 in RAW/LR5 and Cos-7 cells did not induce actin polymerization or protrusion suggesting thatt membrane recruitment was insufficient for WAVE2 regulation. Altogether, these data suggest that IRSp53 links Rac1 to WAVE2 in vivo and its function is crucial for CSF-1-induced F-actin rich protrusions and cell migration in macrophages. This study indicates that Rac1, along with IRSp53 and Abi1, is involved in a more complex and tight regulation of WAVE2 than solely through membrane localization. PMID:18198193

NKG2D Ligands in Tumor Immunity: Two Sides of a Coin.

PubMed

Zhang, Jinyu; Basher, Fahmin; Wu, Jennifer D

2015-01-01

The activating/co-stimulatory receptor NKG2D (natural-killer group 2, member D) is expressed on the surface of all human NK, NKT, CD8(+) T, and subsets of γδ(+) T cells. The significance of NKG2D function in tumor immunity has been well demonstrated in experimental animal models. However, the role of human NKG2D ligands in regulating tumor immunity and cancer prognosis had been controversial in the literature. In this review, we summarize the latest advancement, discuss the controversies, and present evidence that membrane-bound and soluble NKG2D ligands oppositely regulate tumor immunity. We also discuss new perspectives of targeting NKG2D ligands for cancer immunotherapy.

Ectopic adenine nucleotide translocase activity controls extracellular ADP levels and regulates the F1-ATPase-mediated HDL endocytosis pathway on hepatocytes.

PubMed

Cardouat, G; Duparc, T; Fried, S; Perret, B; Najib, S; Martinez, L O

2017-09-01

Ecto-F 1 -ATPase is a complex related to mitochondrial ATP synthase which has been identified as a plasma membrane receptor for apolipoprotein A-I (apoA-I), the major protein of high-density lipoprotein (HDL), and has been shown to contribute to HDL endocytosis in several cell types. On hepatocytes, apoA-I binding to ecto-F 1 -ATPase stimulates extracellular ATP hydrolysis into ADP, which subsequently activates a P2Y 13 -mediated HDL endocytosis pathway. Interestingly, other mitochondrial proteins have been found to be expressed at the plasma membrane of several cell types. Among these, adenine nucleotide translocase (ANT) is an ADP/ATP carrier but its role in controlling extracellular ADP levels and F 1 -ATPase-mediated HDL endocytosis has never been investigated. Here we confirmed the presence of ANT at the plasma membrane of human hepatocytes. We then showed that ecto-ANT activity increases or reduces extracellular ADP level, depending on the extracellular ADP/ATP ratio. Interestingly, ecto-ANT co-localized with ecto-F 1 -ATPase at the hepatocyte plasma membrane and pharmacological inhibition of ecto-ANT activity increased extracellular ADP level when ecto-F 1 -ATPase was activated by apoA-I. This increase in the bioavailability of extracellular ADP accordingly translated into an increase of HDL endocytosis on human hepatocytes. This study thus uncovered a new location and function of ANT for which activity at the cell surface of hepatocytes modulates the concentration of extracellular ADP and regulates HDL endocytosis. Copyright © 2017. Published by Elsevier B.V.

Fast and facile fabrication of antifouling and hemocompatible PVDF membrane tethered with amino-acid modified PEG film

NASA Astrophysics Data System (ADS)

Zhang, Shuyou; Cao, Jingjing; Ma, Na; You, Meng; Wang, Xushan; Meng, Jianqiang

2018-01-01

A fast and facile protocol is reported aiming at improving the antifouling property and hemocompatibility of poly(vinylidene fluoride) (PVDF) membranes by tethering PEG hydrogel and zwitterion immobilization. The coated PEG hydrogel was first prepared by interfacial polymerization and tethered on an alkali treated PVDF membrane (PVDFA) surface via a simultaneous thio-ene and thiol-epoxy reaction. Then, the thiol groups of cysteine reacted with the epoxy groups in PEG hydrogel to fabricate the PVDFA-g-Cys membrane. The membrane fabrication was complete within less than 20 min and was conducted in mild conditions. The successful preparation of PVDFA-g-Cys membrane was confirmed by ATR-FTIR and XPS. Raman spectroscopy showed that the hydrogels covalently bonded to the PVDF membrane surface. The membrane retained its mechanical strength after modification. The SEM measurements suggested that the membrane became denser after hydrogel coating, meanwhile, the EDX test verified that the functional species uniformly distributed in the membrane matrix. Water contact angle (WCA), protein adsorption and protein filtration tests showed significant improvements in hydrophilicity and antifouling properties for the modified membrane. The negativity of the membrane surface measured by the streaming potential method provides a basis for protein resistance and hemocompatibility. Moreover, the suppressed platelet adhesion and prolonged plasma coagulant time show that the PVDFA-g-Cys membrane has ultralow thrombotic potential and better hemocompatibility. The reported surface modification method combing thio-ene and thio-epoxy chemistry not only facilitates fabrication of hemocompatible PVDF membrane but also provide an universal chemical platform for multifunctionalization of porous membranes.

Shaping off-axis metallic membrane reflectors using optimal boundary shapes and inelastic strains

NASA Technical Reports Server (NTRS)

White, C. V.; Dragovan, M.

2004-01-01

This paper will describe a novel concept for constructing off-axis membrane reflector surfaces. Membrane reflectors have been extensively studied, including investigations into inflated lenticular architectures, shaping by spin casting, shaping using electrostatic forces, and shaping by evacuating behind a membrane surface stretched between circular or annular-shaped supports.

Light-induced modification of plant plasma membrane ion transport.

PubMed

Marten, I; Deeken, R; Hedrich, R; Roelfsema, M R G

2010-09-01

Light is not only the driving force for electron and ion transport in the thylakoid membrane, but also regulates ion transport in various other membranes of plant cells. Light-dependent changes in ion transport at the plasma membrane and associated membrane potential changes have been studied intensively over the last century. These studies, with various species and cell types, revealed that apart from regulation by chloroplasts, plasma membrane transport can be controlled by phytochromes, phototropins or channel rhodopsins. In this review, we compare light-dependent plasma membrane responses of unicellular algae (Eremosphaera and Chlamydomonas), with those of a multicellular alga (Chara), liverworts (Conocephalum), mosses (Physcomitrella) and several angiosperm cell types. Light-dependent plasma membrane responses of Eremosphaera and Chara are characterised by the dominant role of K(+) channels during membrane potential changes. In most other species, the Ca(2+)-dependent activation of plasma membrane anion channels represents a general light-triggered event. Cell type-specific responses are likely to have evolved by modification of this general response or through the development of additional light-dependent signalling pathways. Future research to elucidate these light-activated signalling chains is likely to benefit from the recent identification of S-type anion channel genes and proteins capable of regulating these channels.

Triclosan-immobilized polyamide thin film composite membranes with enhanced biofouling resistance

NASA Astrophysics Data System (ADS)

Park, Sang-Hee; Hwang, Seon Oh; Kim, Taek-Seung; Cho, Arah; Kwon, Soon Jin; Kim, Kyoung Taek; Park, Hee-Deung; Lee, Jung-Hyun

2018-06-01

We report on a strategy to improve biofouling resistance of a polyamide (PA) thin-film composite (TFC) reverse osmosis (RO) membrane via chemically immobilizing triclosan (TC), known as a common organic biocide, on its surface. To facilitate covalent attachment of TC on the membrane surface, TC was functionalized with amine moiety to prepare aminopropyl TC. Then, the TC-immobilized TFC (TFC-TC) membranes were fabricated through a one-step amide formation reaction between amine groups of aminopropyl TC and acyl chloride groups present on the PA membrane surface, which was confirmed by high-resolution XPS. Strong stability of the immobilized TC was also confirmed by a hydraulic washing test. Although the TFC-TC membrane showed slightly reduced separation performance compared to the pristine control, it still maintained a satisfactory RO performance level. Importantly, the TFC-TC membrane exhibited excellent antibacterial activity against both gram negative (E. coli and P. aeruginosa) and gram positive (S. aureus) bacteria along with greatly enhanced resistance to biofilm formation. Our immobilization approach offers a robust and relatively benign strategy to control biofouling of functional surfaces, films and membranes.

Polyester polymer alloy as a high-performance membrane.

PubMed

Igoshi, Tadaaki; Tomisawa, Narumi; Hori, Yoshinori; Jinbo, Yoichi

2011-01-01

Polyester polymer alloy (PEPA) membrane is developed as a synthetic polymermembrane. It consists of two polymers - polyethersulfone (PES) and polyarylate (PAR).The pore size in membrane can be controlled by a blend ratio of PES and PAR. One unique characteristic is that PEPA membrane has three layers of a skin layer on the inner surface, a porous layer in the membrane, and a skin layer on the outer surface, respectively. The permeability of water and substances is controlled by the skin layer on the inner surface. PEPA membrane dialyzer can be adequately considered as a high-performance dialyzer. Furthermore, the skin layer on the outer surface can block endotoxin from the dialysis fluid side. PEPA membrane can therefore be used as an endotoxin-retentive filter. The other unique characteristic is that each amount of albumin loss or β2-microglobulin removal can be controlled by an additive amount of polyvinylpyrrolidone. This means that the PEPA dialyzer can be clinically used to meet the conditions of the patient. Copyright © 2011 S. Karger AG, Basel.