Cavin Family: New Players in the Biology of Caveolae.

PubMed

Nassar, Zeyad D; Parat, Marie-Odile

2015-01-01

Caveolae are specialized small plasma-membrane invaginations that play crucial cellular functions. Two essential protein families are required for caveola formation: membrane caveolin proteins and cytoplasmic cavin proteins. Each family includes members with specific tissue distribution, and their expression is altered under physiological and pathological conditions, implying highly specialized functions. Cavins not only stabilize caveolae, but modulate their morphology and functions as well. Before association with the plasma membrane, cavins form homo- and hetero-oligomers with strikingly strict stoichiometry in the cytosol. At the plasma membrane, they provide an outer peripheral cytosolic layer, necessary for caveola stability. Interestingly, upon stimulation, cavins can be released from caveolae into the cytoplasm in distinct subcomplexes, providing a rapid dynamic link between caveolae and cellular organelles including the nucleus. In this review, we detail the biology of cavins, their structural and functional roles, and their implication in pathophysiology. Copyright © 2015 Elsevier Inc. All rights reserved.

Systems and methods for selective hydrogen transport and measurement

DOEpatents

Glatzmaier, Gregory C

2013-10-29

Systems and methods for selectively removing hydrogen gas from a hydrogen-containing fluid volume are disclosed. An exemplary system includes a proton exchange membrane (PEM) selectively permeable to hydrogen by exclusively conducting hydrogen ions. The system also includes metal deposited as layers onto opposite sides or faces of the PEM to form a membrane-electrode assembly (MEA), each layer functioning as an electrode so that the MEA functions as an electrochemical cell in which the ionic conductors are hydrogen ions, and the MEA functioning as a hydrogen selective membrane (HSM) when located at the boundary between a hydrogen-containing fluid volume and a second fluid.

Uncovering homo-and hetero-interactions on the cell membrane using single particle tracking approaches

NASA Astrophysics Data System (ADS)

Torreno-Pina, Juan A.; Manzo, Carlo; Garcia-Parajo, Maria F.

2016-03-01

The plasma membrane of eukaryotic cells is responsible for a myriad of functions that regulate cell physiology and plays a crucial role in a multitude of processes that include adhesion, migration, signaling recognition and cell-cell communication. This is accomplished by specific interactions between different membrane components such as lipids and proteins on the lipid bilayer but also through interactions with the underlying cortical actin cytoskeleton on the intracellular side and the glycocalyx matrix in close proximity to the extracellular side. Advanced biophysical techniques, including single particle tracking (SPT) have revealed that the lateral diffusion of molecular components on the plasma membrane represents a landmark manifestation of such interactions. Indeed, by studying changes in the diffusivity of individual membrane molecules, including sub-diffusion, confined diffusion and/or transient arrest of molecules in membrane compartments, it has been possible to gain insight on the nature of molecular interactions and to infer on its functional role for cell response. In this review, we will revise some exciting results where SPT has been crucial to reveal homo- and hetero-interactions on the cell membrane.

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.

Active Fragments from Pro- and Antiapoptotic BCL-2 Proteins Have Distinct Membrane Behavior Reflecting Their Functional Divergence

PubMed Central

Guillemin, Yannis; Lopez, Jonathan; Gimenez, Diana; Fuertes, Gustavo; Valero, Juan Garcia; Blum, Loïc; Gonzalo, Philippe; Salgado, Jesùs; Girard-Egrot, Agnès; Aouacheria, Abdel

2010-01-01

Background The BCL-2 family of proteins includes pro- and antiapoptotic members acting by controlling the permeabilization of mitochondria. Although the association of these proteins with the outer mitochondrial membrane is crucial for their function, little is known about the characteristics of this interaction. Methodology/Principal Findings Here, we followed a reductionist approach to clarify to what extent membrane-active regions of homologous BCL-2 family proteins contribute to their functional divergence. Using isolated mitochondria as well as model lipid Langmuir monolayers coupled with Brewster Angle Microscopy, we explored systematically and comparatively the membrane activity and membrane-peptide interactions of fragments derived from the central helical hairpin of BAX, BCL-xL and BID. The results show a connection between the differing abilities of the assayed peptide fragments to contact, insert, destabilize and porate membranes and the activity of their cognate proteins in programmed cell death. Conclusion/Significance BCL-2 family-derived pore-forming helices thus represent structurally analogous, but functionally dissimilar membrane domains. PMID:20140092

Dynamic nuclear polarization methods in solids and solutions to explore membrane proteins and membrane systems.

PubMed

Cheng, Chi-Yuan; Han, Songi

2013-01-01

Membrane proteins regulate vital cellular processes, including signaling, ion transport, and vesicular trafficking. Obtaining experimental access to their structures, conformational fluctuations, orientations, locations, and hydration in membrane environments, as well as the lipid membrane properties, is critical to understanding their functions. Dynamic nuclear polarization (DNP) of frozen solids can dramatically boost the sensitivity of current solid-state nuclear magnetic resonance tools to enhance access to membrane protein structures in native membrane environments. Overhauser DNP in the solution state can map out the local and site-specific hydration dynamics landscape of membrane proteins and lipid membranes, critically complementing the structural and dynamics information obtained by electron paramagnetic resonance spectroscopy. Here, we provide an overview of how DNP methods in solids and solutions can significantly increase our understanding of membrane protein structures, dynamics, functions, and hydration in complex biological membrane environments.

Endoplasmic Reticulum - Plasma Membrane Crosstalk Mediated by the Extended Synaptotagmins.

PubMed

Saheki, Yasunori

2017-01-01

The endoplasmic reticulum (ER) possesses multiplicity of functions including protein synthesis, membrane lipid biogenesis, and Ca 2+ storage and has broad localization throughout the cell. While the ER and most other membranous organelles are highly interconnected via vesicular traffic that relies on membrane budding and fusion reactions, the ER forms direct contacts with virtually all other membranous organelles, including the plasma membrane (PM), without membrane fusion. Growing evidence suggests that these contacts play major roles in cellular physiology, including the regulation of Ca 2+ homeostasis and signaling and control of cellular lipid homeostasis. Extended synaptotagmins (E-Syts) are evolutionarily conserved family of ER-anchored proteins that tether the ER to the PM in PM PI(4,5)P 2 -dependent and cytosolic Ca 2+ -regulated manner. In addition, E-Syts possess a cytosolically exposed lipid-harboring module that confers the ability to transfer/exchange glycerolipids between the ER and the PM at E-Syts-mediated ER-PM contacts. In this chapter, the functions of ER-PM contacts and their role in non-vesicular lipid transport with special emphasis on the crosstalk between the two bilayers mediated by E-Syts will be discussed.

An Extended Proteome Map of the Lysosomal Membrane Reveals Novel Potential Transporters*

PubMed Central

Chapel, Agnès; Kieffer-Jaquinod, Sylvie; Sagné, Corinne; Verdon, Quentin; Ivaldi, Corinne; Mellal, Mourad; Thirion, Jaqueline; Jadot, Michel; Bruley, Christophe; Garin, Jérôme; Gasnier, Bruno; Journet, Agnès

2013-01-01

Lysosomes are membrane-bound endocytic organelles that play a major role in degrading cell macromolecules and recycling their building blocks. A comprehensive knowledge of the lysosome function requires an extensive description of its content, an issue partially addressed by previous proteomic analyses. However, the proteins underlying many lysosomal membrane functions, including numerous membrane transporters, remain unidentified. We performed a comparative, semi-quantitative proteomic analysis of rat liver lysosome-enriched and lysosome-nonenriched membranes and used spectral counts to evaluate the relative abundance of proteins. Among a total of 2,385 identified proteins, 734 proteins were significantly enriched in the lysosomal fraction, including 207 proteins already known or predicted as endo-lysosomal and 94 proteins without any known or predicted subcellular localization. The remaining 433 proteins had been previously assigned to other subcellular compartments but may in fact reside on lysosomes either predominantly or as a secondary location. Many membrane-associated complexes implicated in diverse processes such as degradation, membrane trafficking, lysosome biogenesis, lysosome acidification, signaling, and nutrient sensing were enriched in the lysosomal fraction. They were identified to an unprecedented extent as most, if not all, of their subunits were found and retained by our screen. Numerous transporters were also identified, including 46 novel potentially lysosomal proteins. We expressed 12 candidates in HeLa cells and observed that most of them colocalized with the lysosomal marker LAMP1, thus confirming their lysosomal residency. This list of candidate lysosomal proteins substantially increases our knowledge of the lysosomal membrane and provides a basis for further characterization of lysosomal functions. PMID:23436907

Plants and fungi in the era of heterogeneous plasma membranes.

PubMed

Opekarová, M; Malinsky, J; Tanner, W

2010-09-01

Examples from yeast and plant cells are described that show that their plasma membrane is laterally compartmented. Distinct lateral domains encompassing both specific lipids and integral proteins coexist within the plane of the plasma membrane. The compartments are either spatially stable and include distinct sets of proteins, or they are transiently formed to accomplish diverse functions. They are not related to lipid rafts or their clusters, as defined for mammalian cells. This review summarises only well-documented compartments of plasma membranes from plants and fungi, which have been recognised using microscopic approaches. In several cases, physiological functions of the membrane compartmentation are revealed.

Oligomerization but Not Membrane Bending Underlies the Function of Certain F-BAR Proteins in Cell Motility and Cytokinesis.

PubMed

McDonald, Nathan A; Vander Kooi, Craig W; Ohi, Melanie D; Gould, Kathleen L

2015-12-21

F-BAR proteins function in diverse cellular processes by linking membranes to the actin cytoskeleton. Through oligomerization, multiple F-BAR domains can bend membranes into tubules, though the physiological importance of F-BAR-to-F-BAR assemblies is not yet known. Here, we investigate the F-BAR domain of the essential cytokinetic scaffold, Schizosaccharomyces pombe Cdc15, during cytokinesis. Challenging a widely held view that membrane deformation is a fundamental property of F-BARs, we report that the Cdc15 F-BAR binds, but does not deform, membranes in vivo or in vitro, and six human F-BAR domains-including those from Fer and RhoGAP4-share this property. Nevertheless, tip-to-tip interactions between F-BAR dimers are critical for Cdc15 oligomerization and high-avidity membrane binding, stabilization of contractile ring components at the medial cortex, and the fidelity of cytokinesis. F-BAR oligomerization is also critical for Fer and RhoGAP4 physiological function, demonstrating its broad importance to F-BAR proteins that function without membrane bending. Copyright © 2015 Elsevier Inc. All rights reserved.

Hunting for low abundant redox proteins in plant plasma membranes.

PubMed

Lüthje, Sabine; Hopff, David; Schmitt, Anna; Meisrimler, Claudia-Nicole; Menckhoff, Ljiljana

2009-04-13

Nowadays electron transport (redox) systems in plasma membranes appear well established. Members of the flavocytochrome b family have been identified by their nucleotide acid sequences and characterized on the transcriptional level. For their gene products functions have been demonstrated in iron uptake and oxidative stress including biotic interactions, abiotic stress factors and plant development. In addition, NAD(P)H-dependent oxidoreductases and b-type cytochromes have been purified and characterized from plasma membranes. Several of these proteins seem to belong to the group of hypothetical or unknown proteins. Low abundance and the lack of amino acid sequence data for these proteins still hamper their functional analysis. Consequently, little is known about the physiological function and regulation of these enzymes. In recent years evidence has been presented for the existence of microdomains (so-called lipid rafts) in plasma membranes and their interaction with specific membrane proteins. The identification of redox systems in detergent insoluble membranes supports the idea that redox systems may have important functions in signal transduction, stress responses, cell wall metabolism, and transport processes. This review summarizes our present knowledge on plasma membrane redox proteins and discusses alternative strategies to investigate the function and regulation of these enzymes.

Outer membrane proteins of pathogenic spirochetes

PubMed Central

Cullen, Paul A.; Haake, David A.; Adler, Ben

2009-01-01

Pathogenic spirochetes are the causative agents of several important diseases including syphilis, Lyme disease, leptospirosis, swine dysentery, periodontal disease and some forms of relapsing fever. Spirochetal bacteria possess two membranes and the proteins present in the outer membrane are at the site of interaction with host tissue and the immune system. This review describes the current knowledge in the field of spirochetal outer membrane protein (OMP) biology. What is known concerning biogenesis and structure of OMPs, with particular regard to the atypical signal peptide cleavage sites observed amongst the spirochetes, is discussed. We examine the functions that have been determined for several spirochetal OMPs including those that have been demonstrated to function as adhesins, porins or to have roles in complement resistance. A detailed description of the role of spirochetal OMPs in immunity, including those that stimulate protective immunity or that are involved in antigenic variation, is given. A final section is included which covers experimental considerations in spirochetal outer membrane biology. This section covers contentious issues concerning cellular localization of putative OMPs, including determination of surface exposure. A more detailed knowledge of spirochetal OMP biology will hopefully lead to the design of new vaccines and a better understanding of spirochetal pathogenesis. PMID:15449605

The Plasma Membrane Calcium Pump

NASA Technical Reports Server (NTRS)

Rasmussen, H.

1983-01-01

Three aspect of cellular calcium metabolism in animal cells was discussed including the importance of the plasma membrane in calcium homeostasis, experiments dealing with the actual mechanism of the calcium pump, and the function of the pump in relationship to the mitochondria and to the function of calmodulin in the intact cell.

Membrane Lipid Replacement for chronic illnesses, aging and cancer using oral glycerolphospholipid formulations with fructooligosaccharides to restore phospholipid function in cellular membranes, organelles, cells and tissues.

PubMed

Nicolson, Garth L; Ash, Michael E

2017-09-01

Membrane Lipid Replacement is the use of functional, oral supplements containing mixtures of cell membrane glycerolphospholipids, plus fructooligosaccharides (for protection against oxidative, bile acid and enzymatic damage) and antioxidants, in order to safely replace damaged, oxidized, membrane phospholipids and restore membrane, organelle, cellular and organ function. Defects in cellular and intracellular membranes are characteristic of all chronic medical conditions, including cancer, and normal processes, such as aging. Once the replacement glycerolphospholipids have been ingested, dispersed, complexed and transported, while being protected by fructooligosaccharides and several natural mechanisms, they can be inserted into cell membranes, lipoproteins, lipid globules, lipid droplets, liposomes and other carriers. They are conveyed by the lymphatics and blood circulation to cellular sites where they are endocytosed or incorporated into or transported by cell membranes. Inside cells the glycerolphospholipids can be transferred to various intracellular membranes by lipid globules, liposomes, membrane-membrane contact or by lipid carrier transfer. Eventually they arrive at their membrane destinations due to 'bulk flow' principles, and there they can stimulate the natural removal and replacement of damaged membrane lipids while undergoing further enzymatic alterations. Clinical trials have shown the benefits of Membrane Lipid Replacement in restoring mitochondrial function and reducing fatigue in aged subjects and chronically ill patients. Recently Membrane Lipid Replacement has been used to reduce pain and other symptoms as well as removing hydrophobic chemical contaminants, suggesting that there are additional new uses for this safe, natural medicine supplement. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions

PubMed Central

Schwechheimer, Carmen; Kuehn, Meta J.

2017-01-01

Outer-membrane vesicles (OMVs) are spherical buds of the outer membrane filled with periplasmic content and are commonly produced by Gram-negative bacteria. The production of OMVs allows bacteria to interact with their environment, and OMVs have been found to mediate diverse functions, including promoting pathogenesis, enabling bacterial survival during stress conditions and regulating microbial interactions within bacterial communities. Additionally, because of this functional versatility, researchers have begun to explore OMVs as a platform for bioengineering applications. In this Review, we discuss recent advances in the study of OMVs, focusing on new insights into the mechanisms of biogenesis and the functions of these vesicles. PMID:26373371

A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria

PubMed Central

Hoppins, Suzanne; Collins, Sean R.; Cassidy-Stone, Ann; Hummel, Eric; DeVay, Rachel M.; Lackner, Laura L.; Westermann, Benedikt; Schuldiner, Maya

2011-01-01

To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane–associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria. PMID:21987634

Synthetic Molecular Evolution of Membrane-Active Peptides

NASA Astrophysics Data System (ADS)

Wimley, William

The physical chemistry of membrane partitioning largely determines the function of membrane active peptides. Membrane-active peptides have potential utility in many areas, including in the cellular delivery of polar compounds, cancer therapy, biosensor design, and in antibacterial, antiviral and antifungal therapies. Yet, despite decades of research on thousands of known examples, useful sequence-structure-function relationships are essentially unknown. Because peptide-membrane interactions within the highly fluid bilayer are dynamic and heterogeneous, accounts of mechanism are necessarily vague and descriptive, and have little predictive power. This creates a significant roadblock to advances in the field. We are bypassing that roadblock with synthetic molecular evolution: iterative peptide library design and orthogonal high-throughput screening. We start with template sequences that have at least some useful activity, and create small, focused libraries using structural and biophysical principles to design the sequence space around the template. Orthogonal high-throughput screening is used to identify gain-of-function peptides by simultaneously selecting for several different properties (e.g. solubility, activity and toxicity). Multiple generations of iterative library design and screening have enabled the identification of membrane-active sequences with heretofore unknown properties, including clinically relevant, broad-spectrum activity against drug-resistant bacteria and enveloped viruses as well as pH-triggered macromolecular poration.

Erythrocyte membrane-camouflaged polymeric nanoparticles as a biomimetic delivery platform

PubMed Central

Hu, Che-Ming J.; Zhang, Li; Aryal, Santosh; Cheung, Connie; Fang, Ronnie H.; Zhang, Liangfang

2011-01-01

Efforts to extend nanoparticle residence time in vivo have inspired many strategies in particle surface modifications to bypass macrophage uptake and systemic clearance. Here we report a top-down biomimetic approach in particle functionalization by coating biodegradable polymeric nanoparticles with natural erythrocyte membranes, including both membrane lipids and associated membrane proteins for long-circulating cargo delivery. The structure, size and surface zeta potential, and protein contents of the erythrocyte membrane-coated nanoparticles were verified using transmission electron microscopy, dynamic light scattering, and gel electrophoresis, respectively. Mice injections with fluorophore-loaded nanoparticles revealed superior circulation half-life by the erythrocyte-mimicking nanoparticles as compared to control particles coated with the state-of-the-art synthetic stealth materials. Biodistribution study revealed significant particle retention in the blood 72 h following the particle injection. The translocation of natural cellular membranes, their associated proteins, and the corresponding functionalities to the surface of synthetic particles represents a unique approach in nanoparticle functionalization. PMID:21690347

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

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

PubMed

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

2015-05-09

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

Effects of rutin on the physicochemical properties of skin fibroblasts membrane disruption following UV radiation.

PubMed

Dobrzyńska, Izabela; Gęgotek, Agnieszka; Gajko, Ewelina; Skrzydlewska, Elżbieta; Figaszewski, Zbigniew A

2018-02-25

Human skin provides the body's first line of defense against physical and environmental assaults. This study sought to determine how rutin affects the membrane electrical properties, sialic acid content, and lipid peroxidation levels of fibroblast membranes after disruption by ultraviolet (UV) radiation. Changes in cell function may affect the basal electrical surface properties of cell membranes, and changes can be detected by electrokinetic measurements. The charge density of the fibroblast membrane surface was measured as a function of pH. A four-component equilibrium model was used to describe the interaction between ions in solution and ions on the membrane surface. Agreement was found between experimental and theoretical charge variation curves of fibroblast cells between pH 2.5 and 8. Sialic acid content was determined by Svennerholm's resorcinol method, and lipid peroxidation was estimated by measuring the malondialdehyde level. Compared to untreated cells, ultraviolet A (UVA)- or ultraviolet B (UVB)-treated skin cell membranes exhibited higher concentrations of acidic functional groups and higher average association constants with hydroxyl ions, but lower average association constants with hydrogen ions. Moreover, our results showed that UVA and UVB radiation is associated with increased levels of sialic acid and lipid peroxidation products in fibroblasts. Rutin protected cells from some deleterious UV-associated membrane changes, including changes in electrical properties, oxidative state, and biological functions. Copyright © 2018 Elsevier B.V. All rights reserved.

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

PubMed Central

Butler, Emily K.; Davis, Rebecca M.; Bari, Vase; Nicholson, Paul A.

2013-01-01

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis. PMID:23935042

Functional imaging of microdomains in cell membranes.

PubMed

Duggan, James; Jamal, Ghadir; Tilley, Mark; Davis, Ben; McKenzie, Graeme; Vere, Kelly; Somekh, Michael G; O'Shea, Paul; Harris, Helen

2008-10-01

The presence of microdomains or rafts within cell membranes is a topic of intense study and debate. The role of these structures in cell physiology, however, is also not yet fully understood with many outstanding problems. This problem is partly based on the small size of raft structures that presents significant problems to their in vivo study, i.e., within live cell membranes. But the structure and dynamics as well as the factors that control the assembly and disassembly of rafts are also of major interest. In this review we outline some of the problems that the study of rafts in cell membranes present as well as describing some views of what are considered the generalised functions of membrane rafts. We point to the possibility that there may be several different 'types' of membrane raft in cell membranes and consider the factors that affect raft assembly and disassembly, particularly, as some researchers suggest that the lifetimes of rafts in cell membranes may be sub-second. We attempt to review some of the methods that offer the ability to interrogate rafts directly as well as describing factors that appear to affect their functionality. The former include both near-field and far-field optical approaches as well as scanning probe techniques. Some of the advantages and disadvantages of these techniques are outlined. Finally, we describe our own views of raft functionality and properties, particularly, concerning the membrane dipole potential, and describe briefly some of the imaging strategies we have developed for their study.

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

PubMed

Kessels, Michael M; Qualmann, Britta

2015-09-01

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

Vibrational Spectroscopy as a Promising Toolbox for Analyzing Functionalized Ceramic Membranes.

PubMed

Kiefer, Johannes; Bartels, Julia; Kroll, Stephen; Rezwan, Kurosch

2018-01-01

Ceramic materials find use in many fields including the life sciences and environmental engineering. For example, ceramic membranes have shown to be promising filters for water treatment and virus retention. The analysis of such materials, however, remains challenging. In the present study, the potential of three vibrational spectroscopic methods for characterizing functionalized ceramic membranes for water treatment is evaluated. For this purpose, Raman scattering, infrared (IR) absorption, and solvent infrared spectroscopy (SIRS) were employed. The data were analyzed with respect to spectral changes as well as using principal component analysis (PCA). The Raman spectra allow an unambiguous discrimination of the sample types. The IR spectra do not change systematically with functionalization state of the material. Solvent infrared spectroscopy allows a systematic distinction and enables studying the molecular interactions between the membrane surface and the solvent.

Outer Membrane Targeting of Passenger Proteins by the Vacuolating Cytotoxin Autotransporter of Helicobacter pylori

PubMed Central

Fischer, Wolfgang; Buhrdorf, Renate; Gerland, Elke; Haas, Rainer

2001-01-01

Helicobacter pylori produces a number of proteins associated with the outer membrane, including adhesins and the vacuolating cytotoxin. These proteins are supposed to integrate into the outer membrane by β-barrel structures, characteristic of the family of autotransporter proteins. By using the SOMPES (shuttle vector-based outer membrane protein expression) system for outer membrane protein production, we were able to functionally express in H. pylori the cholera toxin B subunit genetically fused to the C-terminal VacA domain. We demonstrate that the fusion protein is translocated to the H. pylori outer membrane and that the CtxB domain is exposed on the H. pylori surface. Thus, we provide the first experimental evidence that the C-terminal β-domain of VacA can transport a foreign passenger protein to the H. pylori surface and hence acts as a functional autotransporter. PMID:11598049

Proteome analysis of the triton-insoluble erythrocyte membrane skeleton.

PubMed

Basu, Avik; Harper, Sandra; Pesciotta, Esther N; Speicher, Kaye D; Chakrabarti, Abhijit; Speicher, David W

2015-10-14

Erythrocyte shape and membrane integrity is imparted by the membrane skeleton, which can be isolated as a Triton X-100 insoluble structure that retains the biconcave shape of intact erythrocytes, indicating isolation of essentially intact membrane skeletons. These erythrocyte "Triton Skeletons" have been studied morphologically and biochemically, but unbiased proteome analysis of this substructure of the membrane has not been reported. In this study, different extraction buffers and in-depth proteome analyses were used to more fully define the protein composition of this functionally critical macromolecular complex. As expected, the major, well-characterized membrane skeleton proteins and their associated membrane anchors were recovered in good yield. But surprisingly, a substantial number of additional proteins that are not considered in erythrocyte membrane skeleton models were recovered in high yields, including myosin-9, lipid raft proteins (stomatin, flotillin1 and 2), multiple chaperone proteins (HSPs, protein disulfide isomerase and calnexin), and several other proteins. These results show that the membrane skeleton is substantially more complex than previous biochemical studies indicated, and it apparently has localized regions with unique protein compositions and functions. This comprehensive catalog of the membrane skeleton should lead to new insights into erythrocyte membrane biology and pathogenic mutations that perturb membrane stability. Biological significance Current models of erythrocyte membranes describe fairly simple homogenous structures that are incomplete. Proteome analysis of the erythrocyte membrane skeleton shows that it is quite complex and includes a substantial number of proteins whose roles and locations in the membrane are not well defined. Further elucidation of interactions involving these proteins and definition of microdomains in the membrane that contain these proteins should yield novel insights into how the membrane skeleton produces the normal biconcave erythrocyte shape and how it is perturbed in pathological conditions that destabilize the membrane. Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of Vanadium-Containing Compounds on Membrane Lipids and on Microdomains Used in Receptor-Mediated Signaling

PubMed Central

Roess, Deborah A.; Smith, Steven M. L.; Winter, Peter; Zhou, Jun; Dou, Ping; Baruah, Bharat; Trujillo, Alejandro M.; Levinger, Nancy E.; Yang, Xioda; Barisas, B. George; Crans, Debbie C.

2011-01-01

There is increasing evidence for the involvement of plasma membrane microdomains in insulin receptor function. Moreover, disruption of these structures, which are typically enriched in sphingomyelin and cholesterol, results in insulin resistance. Treatment strategies for insulin resistance include the use of vanadium compounds which have been shown in animal models to enhance insulin responsiveness. One possible mechanism for insulin-enhancing effects might involve direct effects of vanadium compounds on membrane lipid organization. These changes in lipid organization promote the partitioning of insulin receptors and other receptors into membrane microdomains where receptors are optimally functional. To explore this possibility, we have used several strategies involving vanadium complexes such as [VO2dipic]− (pyridin-2,6-dicarboxylatodioxovanadium(V)), decavanadate (V10O286−, V10), BMOV (bis(maltolato)oxovanadium(IV)) and [VO(saltris)]2 (2-salicylideniminato-2-(hydroxymethyl)-1,3-dihydroxypropane-oxovanadium(V)). Our strategies include an evaluation of interactions between vanadium-containing compounds and model lipid systems, an evaluation of the effects of vanadium compounds on lipid fluidity in erythrocyte membranes, and studies of the effects of vanadium-containing compounds on signaling events initiated by receptors known to use membrane microdomains as signaling platforms. PMID:18729092

Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria

PubMed Central

Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

2014-01-01

Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors. PMID:24808904

Efficient Exploration of Membrane-Associated Phenomena at Atomic Resolution.

PubMed

Vermaas, Josh V; Baylon, Javier L; Arcario, Mark J; Muller, Melanie P; Wu, Zhe; Pogorelov, Taras V; Tajkhorshid, Emad

2015-06-01

Biological membranes constitute a critical component in all living cells. In addition to providing a conducive environment to a wide range of cellular processes, including transport and signaling, mounting evidence has established active participation of specific lipids in modulating membrane protein function through various mechanisms. Understanding lipid-protein interactions underlying these mechanisms at a sufficiently high resolution has proven extremely challenging, partly due to the semi-fluid nature of the membrane. In order to address this challenge computationally, multiple methods have been developed, including an alternative membrane representation termed highly mobile membrane mimetic (HMMM) in which lateral lipid diffusion has been significantly enhanced without compromising atomic details. The model allows for efficient sampling of lipid-protein interactions at atomic resolution, thereby significantly enhancing the effectiveness of molecular dynamics simulations in capturing membrane-associated phenomena. In this review, after providing an overview of HMMM model development, we will describe briefly successful application of the model to study a variety of membrane processes, including lipid-dependent binding and insertion of peripheral proteins, the mechanism of phospholipid insertion into lipid bilayers, and characterization of optimal tilt angle of transmembrane helices. We conclude with practical recommendations for proper usage of the model in simulation studies of membrane processes.

Polymeric capsule-cushioned leukocyte cell membrane vesicles as a biomimetic delivery platform

NASA Astrophysics Data System (ADS)

Gao, Changyong; Wu, Zhiguang; Lin, Zhihua; Lin, Xiankun; He, Qiang

2016-02-01

We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural cell membrane camouflaged polymeric multilayer capsules with the immunosuppressive and tumor-recognition functionalities of natural leukocytes provide a new biomimetic delivery platform for disease therapy.We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural cell membrane camouflaged polymeric multilayer capsules with the immunosuppressive and tumor-recognition functionalities of natural leukocytes provide a new biomimetic delivery platform for disease therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08407e

Atomic resolution view into the structure–function relationships of the human myelin peripheral membrane protein P2

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

Ruskamo, Salla; University of Oulu, Oulu; Yadav, Ravi P.

2014-01-01

The structure of the human myelin peripheral membrane protein P2 has been refined at 0.93 Å resolution. In combination with functional experiments in vitro, in vivo and in silico, the fine details of the structure–function relationships in P2 are emerging. P2 is a fatty acid-binding protein expressed in vertebrate peripheral nerve myelin, where it may function in bilayer stacking and lipid transport. P2 binds to phospholipid membranes through its positively charged surface and a hydrophobic tip, and accommodates fatty acids inside its barrel structure. The structure of human P2 refined at the ultrahigh resolution of 0.93 Å allows detailed structuralmore » analyses, including the full organization of an internal hydrogen-bonding network. The orientation of the bound fatty-acid carboxyl group is linked to the protonation states of two coordinating arginine residues. An anion-binding site in the portal region is suggested to be relevant for membrane interactions and conformational changes. When bound to membrane multilayers, P2 has a preferred orientation and is stabilized, and the repeat distance indicates a single layer of P2 between membranes. Simulations show the formation of a double bilayer in the presence of P2, and in cultured cells wild-type P2 induces membrane-domain formation. Here, the most accurate structural and functional view to date on P2, a major component of peripheral nerve myelin, is presented, showing how it can interact with two membranes simultaneously while going through conformational changes at its portal region enabling ligand transfer.« less

Structure and dynamics of cationic membrane peptides and proteins: Insights from solid-state NMR

PubMed Central

Hong, Mei; Su, Yongchao

2011-01-01

Many membrane peptides and protein domains contain functionally important cationic Arg and Lys residues, whose insertion into the hydrophobic interior of the lipid bilayer encounters significant energy barriers. To understand how these cationic molecules overcome the free energy barrier to insert into the lipid membrane, we have used solid-state NMR spectroscopy to determine the membrane-bound topology of these peptides. A versatile array of solid-state NMR experiments now readily yields the conformation, dynamics, orientation, depth of insertion, and site-specific protein–lipid interactions of these molecules. We summarize key findings of several Arg-rich membrane peptides, including β-sheet antimicrobial peptides, unstructured cell-penetrating peptides, and the voltage-sensing helix of voltage-gated potassium channels. Our results indicate the central role of guanidinium-phosphate and guanidinium-water interactions in dictating the structural topology of these cationic molecules in the lipid membrane, which in turn account for the mechanisms of this functionally diverse class of membrane peptides. PMID:21344534

Surface functionalization of a polymeric lipid bilayer for coupling a model biological membrane with molecules, cells, and microstructures.

PubMed

Morigaki, Kenichi; Mizutani, Kazuyuki; Saito, Makoto; Okazaki, Takashi; Nakajima, Yoshihiro; Tatsu, Yoshiro; Imaishi, Hiromasa

2013-02-26

We describe a stable and functional model biological membrane based on a polymerized lipid bilayer with a chemically modified surface. A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing phospholipids, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DiynePE). DiynePC formed a stable bilayer structure, whereas the ethanolamine headgroup of DiynePE enabled functional molecules to be grafted onto the membrane surface. Copolymerization of DiynePC and DiynePE resulted in a robust bilayer. Functionalization of the polymeric bilayer provided a route to a robust and biomimetic surface that can be linked with biomolecules, cells, and three-dimensional (3D) microstructures. Biotin and peptides were grafted onto the polymeric bilayer for attaching streptavidin and cultured mammalian cells by molecular recognition, respectively. Nonspecific adsorption of proteins and cells on polymeric bilayers was minimum. DiynePE was also used to attach a microstructure made of an elastomer (polydimethylsiloxan: PDMS) onto the membrane, forming a confined aqueous solution between the two surfaces. The microcompartment enabled us to assay the activity of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid bilayers were incorporated together with membrane-bound proteins by lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid membrane of functionalized polymeric bilayers and fluid bilayers offers a novel platform for a wide range of biomedical applications including biosensor, bioassay, cell culture, and cell-based assay.

Helix formation and stability in membranes.

PubMed

McKay, Matthew J; Afrose, Fahmida; Koeppe, Roger E; Greathouse, Denise V

2018-02-13

In this article we review current understanding of basic principles for the folding of membrane proteins, focusing on the more abundant alpha-helical class. Membrane proteins, vital to many biological functions and implicated in numerous diseases, fold into their active conformations in the complex environment of the cell bilayer membrane. While many membrane proteins rely on the translocon and chaperone proteins to fold correctly, others can achieve their functional form in the absence of any translation apparatus or other aides. Nevertheless, the spontaneous folding process is not well understood at the molecular level. Recent findings suggest that helix fraying and loop formation may be important for overall structure, dynamics and regulation of function. Several types of membrane helices with ionizable amino acids change their topology with pH. Additionally we note that some peptides, including many that are rich in arginine, and a particular analogue of gramicidin, are able passively to translocate across cell membranes. The findings indicate that a final protein structure in a lipid-bilayer membrane is sequence-based, with lipids contributing to stability and regulation. While much progress has been made toward understanding the folding process for alpha-helical membrane proteins, it remains a work in progress. This article is part of a Special Issue entitled: Emergence of Complex Behavior in Biomembranes edited by Marjorie Longo. Copyright © 2018 Elsevier B.V. All rights reserved.

Membrane protein properties revealed through data-rich electrostatics calculations

PubMed Central

Guerriero, Christopher J.; Brodsky, Jeffrey L.; Grabe, Michael

2015-01-01

SUMMARY The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem including: full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane induced pKa shifts, calculation of non-polar energies, and command-line scripting for large scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane potentially revealing interesting functional information. PMID:26118532

Membrane Protein Properties Revealed through Data-Rich Electrostatics Calculations.

PubMed

Marcoline, Frank V; Bethel, Neville; Guerriero, Christopher J; Brodsky, Jeffrey L; Grabe, Michael

2015-08-04

The electrostatic properties of membrane proteins often reveal many of their key biophysical characteristics, such as ion channel selectivity and the stability of charged membrane-spanning segments. The Poisson-Boltzmann (PB) equation is the gold standard for calculating protein electrostatics, and the software APBSmem enables the solution of the PB equation in the presence of a membrane. Here, we describe significant advances to APBSmem, including full automation of system setup, per-residue energy decomposition, incorporation of PDB2PQR, calculation of membrane-induced pKa shifts, calculation of non-polar energies, and command-line scripting for large-scale calculations. We highlight these new features with calculations carried out on a number of membrane proteins, including the recently solved structure of the ion channel TRPV1 and a large survey of 1,614 membrane proteins of known structure. This survey provides a comprehensive list of residues with large electrostatic penalties for being embedded in the membrane, potentially revealing interesting functional information. Copyright © 2015 Elsevier Ltd. All rights reserved.

Membranes with functionalized carbon nanotube pores for selective transport

DOEpatents

Bakajin, Olgica; Noy, Aleksandr; Fornasiero, Francesco; Park, Hyung Gyu; Holt, Jason K; Kim, Sangil

2015-01-27

Provided herein composition and methods for nanoporous membranes comprising single walled, double walled, or multi-walled carbon nanotubes embedded in a matrix material. Average pore size of the carbon nanotube can be 6 nm or less. These membranes are a robust platform for the study of confined molecular transport, with applications in liquid and gas separations and chemical sensing including desalination, dialysis, and fabric formation.

Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane.

PubMed

Schmidt, Daniel; MacKinnon, Roderick

2008-12-09

Voltage-dependent K(+) (Kv) channels underlie action potentials through gating conformational changes that are driven by membrane voltage. In this study of the paddle chimera Kv channel, we demonstrate that the rate of channel opening, the voltage dependence of the open probability, and the maximum achievable open probability depend on the lipid membrane environment. The activity of the voltage sensor toxin VsTx1, which interferes with voltage-dependent gating by partitioning into the membrane and binding to the channel, also depends on the membrane. Membrane environmental factors that influence channel function are divisible into two general categories: lipid compositional and mechanical state. The mechanical state can have a surprisingly large effect on the function of a voltage-dependent K(+) channel, including its pharmacological interaction with voltage sensor toxins. The dependence of VSTx1 activity on the mechanical state of the membrane leads us to hypothesize that voltage sensor toxins exert their effect by perturbing the interaction forces that exist between the channel and the membrane.

Voltage-dependent K+ channel gating and voltage sensor toxin sensitivity depend on the mechanical state of the lipid membrane

PubMed Central

Schmidt, Daniel; MacKinnon, Roderick

2008-01-01

Voltage-dependent K+ (Kv) channels underlie action potentials through gating conformational changes that are driven by membrane voltage. In this study of the paddle chimera Kv channel, we demonstrate that the rate of channel opening, the voltage dependence of the open probability, and the maximum achievable open probability depend on the lipid membrane environment. The activity of the voltage sensor toxin VsTx1, which interferes with voltage-dependent gating by partitioning into the membrane and binding to the channel, also depends on the membrane. Membrane environmental factors that influence channel function are divisible into two general categories: lipid compositional and mechanical state. The mechanical state can have a surprisingly large effect on the function of a voltage-dependent K+ channel, including its pharmacological interaction with voltage sensor toxins. The dependence of VSTx1 activity on the mechanical state of the membrane leads us to hypothesize that voltage sensor toxins exert their effect by perturbing the interaction forces that exist between the channel and the membrane. PMID:19050073

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids

PubMed Central

Kim, JiHyun; Huang, Zhen; St. Clair, Johnna R.; Brown, Deborah A.; London, Erwin

2016-01-01

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70–80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids. PMID:27872310

Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids.

PubMed

Li, Guangtao; Kim, JiHyun; Huang, Zhen; St Clair, Johnna R; Brown, Deborah A; London, Erwin

2016-12-06

Our understanding of membranes and membrane lipid function has lagged far behind that of nucleic acids and proteins, largely because it is difficult to manipulate cellular membrane lipid composition. To help solve this problem, we show that methyl-α-cyclodextrin (MαCD)-catalyzed lipid exchange can be used to maximally replace the sphingolipids and phospholipids in the outer leaflet of the plasma membrane of living mammalian cells with exogenous lipids, including unnatural lipids. In addition, lipid exchange experiments revealed that 70-80% of cell sphingomyelin resided in the plasma membrane outer leaflet; the asymmetry of metabolically active cells was similar to that previously defined for erythrocytes, as judged by outer leaflet lipid composition; and plasma membrane outer leaflet phosphatidylcholine had a significantly lower level of unsaturation than phosphatidylcholine in the remainder of the cell. The data also provided a rough estimate for the total cellular lipids residing in the plasma membrane (about half). In addition to such lipidomics applications, the exchange method should have wide potential for investigations of lipid function and modification of cellular behavior by modification of lipids.

Gradient composite metal-ceramic foam as supportive component for planar SOFCs and MIEC membranes

NASA Astrophysics Data System (ADS)

Smorygo, Oleg; Mikutski, Vitali; Marukovich, Alexander; Sadykov, Vladislav; Usoltsev, Vladimir; Mezentseva, Natalia; Borodinecs, Anatolijs; Bobrenok, Oleg

2011-06-01

A novel approach to the design of planar gradient porous supports for the thin-film SOFCs and MIEC membranes is described. The support's thermal expansion is controlled by the creation of a two-component composite metal-ceramic foam structure. Thin MIEC membranes and SOFCs were prepared on the composite supports by the layerwise deposition of composite functional layers including complex fluorites and perovskites. Lab-scale studies demonstrated promising performance of both MIEC membrane and SOFC.

Performance of cellulose acetate membrane with different additives for palm oil mill effluent (POME) liquid waste treatment

NASA Astrophysics Data System (ADS)

Aprilia, N. A. S.; Fauzi; Azmi, N.; Najwan, N.; Amin, A.

2018-03-01

Performance of cellulose acetate membrane for treatment of POME liquid has studied with different additives. Cellulose acetate membranes were prepared with different additive ie formamide and polyethylene glycol and used acetone as solvent. The function of formamide and polyethylene glycol (PEG) is to increase the porosity of the membrane surface. Performance of the membrane were included SEM, FT-IR and coefficient permeability. Membrane performance has been performed for percent rejection of total suspended solid (TSS) and turbidity of POME liquid waste. Cellulose acetate with formamide shows an increased percentage of rejection in removing TSS and turbidity than cellulose acetate with PEG.

Basilar-membrane responses to broadband noise modeled using linear filters with rational transfer functions.

PubMed

Recio-Spinoso, Alberto; Fan, Yun-Hui; Ruggero, Mario A

2011-05-01

Basilar-membrane responses to white Gaussian noise were recorded using laser velocimetry at basal sites of the chinchilla cochlea with characteristic frequencies near 10 kHz and first-order Wiener kernels were computed by cross correlation of the stimuli and the responses. The presence or absence of minimum-phase behavior was explored by fitting the kernels with discrete linear filters with rational transfer functions. Excellent fits to the kernels were obtained with filters with transfer functions including zeroes located outside the unit circle, implying nonminimum-phase behavior. These filters accurately predicted basilar-membrane responses to other noise stimuli presented at the same level as the stimulus for the kernel computation. Fits with all-pole and other minimum-phase discrete filters were inferior to fits with nonminimum-phase filters. Minimum-phase functions predicted from the amplitude functions of the Wiener kernels by Hilbert transforms were different from the measured phase curves. These results, which suggest that basilar-membrane responses do not have the minimum-phase property, challenge the validity of models of cochlear processing, which incorporate minimum-phase behavior. © 2011 IEEE

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

PubMed

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

2016-09-01

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

Structural and functional changes in acute liver injury.

PubMed

Smuckler, E A

1976-06-01

Carbon tetrachloride produces liver cell injury in a variety of animal species. The first structurally recognizable changes occur in the endoplasmic reticulum, with alteration in ribosome-membrane interactions. Later there is an increase in intracellular fat, and the formation of tangled nets of the ergastoplasm. At no time are there changes in mitochondria or single membrane limited bodies in cells with intact plasmalemma, although a relative increase in cell sap may appear. In dead cells (those with plasmalemma discontinuties) crystalline deposits of calcium phosphatase may be noted. Functional changes are related to the endoplasmic reticulum and the plasma membrane. An early decrease in protein synthesis takes place; an accumulation of neutral lipid is related to this change. Later alterations in the ergastoplasmic functions (e.g., mixed function oxidation) occurs. Carbon tetrachloride is not the active agent; rather, a product of its metabolism, probably the CC1, free radical, is. The mechanisms of injury include macromolecular adduction and peroxide propagation. A third possibility includes a cascade effect with the production of secondary and tertiary products, also toxic in nature, with the ability to produce more widespread damage to intracellular structures.

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry-Based Approach.

PubMed

Murata, Michio; Sugiyama, Shigeru; Matsuoka, Shigeru; Matsumori, Nobuaki

2015-08-01

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three-dimensional structures. As lipids are dynamic by nature, their "structure" does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid-raft-related molecules, lipid-protein interactions, and membrane-active natural products, we discuss current perspectives on membrane structural biology. © 2015 The Chemical Society of Japan & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of RPE65, CRALBP, VEGF, CD68, and tyrosinase gene expression in human retinal pigment epithelial cells cultured on amniotic membrane.

PubMed

Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Khalooghi, Keynoush; Ahmadieh, Hamid; Kanavi, Mojgan Rezaie; Samiei, Shahram; Pakravesh, Jalil

2011-06-01

The retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina. Tissue engineering using amniotic membrane as a substrate to culture RPE cells may provide a promising new strategy to replace damaged RPE. We established a method of culturing RPE cells over the amniotic membrane as a support for their growth and transplantation. The transcription of specific genes involved in cellular function of native RPE, including RPE65, CRALBP, VEGF, CD68, and tyrosinase, were then measured using quantitative real-time PCR. Data showed a considerable increase in transcription of RPE65, CD68, and VEGF in RPE cells cultured on amniotic membrane. The amounts of CRALBP and tyrosinase transcripts were not affected. This may simply indicate that amniotic membrane restricted dedifferentiation of RPE cells in culture. The results suggest that amniotic membrane may be considered as an elective biological substrate for RPE cell culture.

Functional advantages conferred by extracellular prokaryotic membrane vesicles.

PubMed

Manning, Andrew J; Kuehn, Meta J

2013-01-01

The absence of subcellular organelles is a characteristic typically used to distinguish prokaryotic from eukaryotic cells. But recent discoveries do not support this dogma. Over the past 50 years, researchers have begun to appreciate and characterize Gram-negative bacterial outer membrane-derived vesicles and Gram-positive and archaeal membrane vesicles. These extracellular, membrane-bound organelles can perform a variety of functions, including binding and delivery of DNA, transport of virulence factors, protection of the cell from outer membrane targeting antimicrobials and ridding the cell of toxic envelope proteins. Here, we review the contributions of these extracellular organelles to prokaryotic physiology and compare these with the contributions of the bacterial interior membrane-bound organelles responsible for harvesting light energy and for generating magnetic crystals of heavy metals. Understanding the roles of these multifunctional extracellular vesicle organelles as microbial tools will help us to better realize the diverse interactions that occur in our polymicrobial world. Copyright © 2013 S. Karger AG, Basel.

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.

Modification of Erythrocyte Membrane Fatty Acid Contents After Kidney Transplantation: A Prospective Study.

PubMed

Son, Y K; Kwon, H; Lee, H W; Jeong, E G; Lee, S M; Kim, S E; Park, Y; An, W S

2018-06-01

Modifications of erythrocyte membrane fatty acid (FA) contents may affect cellular function or transmembrane receptors. One cross-sectional study has shown that kidney transplant (KTP) recipients have lower erythrocyte membrane oleic acid content than dialysis patients do. Therefore, we prospectively tested whether erythrocyte membrane contents of FA including oleic acid change after KTP. We recruited 23 KTP recipients (September 2011 through May 2014). Blood samples were obtained immediately before KTP and 6 months after. Erythrocyte membrane FA contents were measured by gas chromatography. Mean age of the enrolled KTP recipients was 45.3 ± 10.9 years, and men represented 66.7% of the cases. ABO-incompatible KTPs constituted 14.3% and cadaver donors 42.9% of the cases. Steroids, mycophenolate mofetil, and tacrolimus were used as immunosuppressive treatment. There was no significant difference in dietary consumption between time points before and 6 months after KTP. Total cholesterol and low-density lipoprotein cholesterol levels were significantly higher at 6 months after KTP as compared with baseline. Erythrocyte membrane contents of polyunsaturated FA, ω-3 FA, ω-6 FA, and the ω-3 index were significantly higher, but erythrocyte membrane contents of total saturated FAs, total monounsaturated FAs, including oleic acid, total trans-FA, palmitoleic acid, and the ω-6-to-ω-3 ratio were significantly lower at 6 months after KTP. Erythrocyte membrane FA contents significantly changed toward a more favorable cardiovascular profile after KTP. These changes in erythrocyte membrane FA contents may be related to improved renal function because of the absence of significant dietary changes. Copyright © 2018 Elsevier Inc. All rights reserved.

Neurologic Outcomes After Extracorporeal Membrane Oxygenation-A Systematic Review.

PubMed

Boyle, Katharine; Felling, Ryan; Yiu, Alvin; Battarjee, Wejdan; Schwartz, Jamie McElrath; Salorio, Cynthia; Bembea, Melania M

2018-06-11

The goal of this systematic review of the literature was to summarize neurologic outcomes following neonatal and pediatric extracorporeal membrane oxygenation. We conducted electronic searches of PubMed, Scopus, Web of Science, CINAHL, Cochrane, and EMBASE. Inclusion criteria included publication dates 2000-2016, patient ages 0-18 years, and use of standardized measures to evaluate outcomes after extracorporeal membrane oxygenation. We identified 3,497 unique citations; 60 full-text articles were included in the final review. Studies evaluated patients with congenital diaphragmatic hernia (7), cardiac disease (8), cardiac arrest (13), and mixed populations (32). Follow-up was conducted at hospital discharge in 10 studies (17%) and at a median of 26 months (interquartile range, 8-61 mo) after extracorporeal membrane oxygenation in 50 studies (83%). We found 55 outcome measures that assessed overall health and function (4), global cognitive ability (7), development (4), motor function (5), adaptive function (2), behavior/mood (6), hearing (2), quality of life (2), school achievement (5), speech and language (6), learning and memory (4), and attention and executive function (8). Overall, 10% to as many as 50% of children scored more than 2 SDS below the population mean on cognitive testing. Behavior problems were identified in 16-46% of children tested, and severe motor impairment was reported in 12% of children. Quality of life of former extracorporeal membrane oxygenation patients evaluated at school age or adolescence ranged from similar to healthy peers, to 31-53% having scores more than 1 SD below the population mean. This systematic review of the literature suggests that children who have undergone extracorporeal membrane oxygenation suffer from a wide range of disabilities. A meta-analysis was not feasible due to heterogeneity in pathologies, outcome measures, and age at follow-up, underscoring the importance of developing and employing a core set of outcomes measures in future extracorporeal membrane oxygenation studies.

Recent Advances of Membrane-Cloaked Nanoplatforms for Biomedical Applications.

PubMed

Ai, Xiangzhao; Hu, Ming; Wang, Zhimin; Zhang, Wenmin; Li, Juan; Yang, Huanghao; Lin, Jun; Xing, Bengang

2018-04-18

In terms of the extremely small size and large specific surface area, nanomaterials often exhibit unusual physical and chemical properties, which have recently attracted considerable attention in bionanotechnology and nanomedicine. Currently, the extensive usage of nanotechnology in medicine holds great potential for precise diagnosis and effective therapeutics of various human diseases in clinical practice. However, a detailed understanding regarding how nanomedicine interacts with the intricate environment in complex living systems remains a pressing and challenging goal. Inspired by the diversified membrane structures and functions of natural prototypes, research activities on biomimetic and bioinspired membranes, especially for those cloaking nanosized platforms, have increased exponentially. By taking advantage of the flexible synthesis and multiple functionality of nanomaterials, a variety of unique nanostructures including inorganic nanocrystals and organic polymers have been widely devised to substantially integrate with intrinsic biomoieties such as lipids, glycans, and even cell and bacteria membrane components, which endow these abiotic nanomaterials with specific biological functionalities for the purpose of detailed investigation of the complicated interactions and activities of nanomedicine in living bodies, including their immune response activation, phagocytosis escape, and subsequent clearance from vascular system. In this review, we summarize the strategies established recently for the development of biomimetic membrane-cloaked nanoplatforms derived from inherent host cells (e.g., erythrocytes, leukocytes, platelets, and exosomes) and invasive pathogens (e.g., bacteria and viruses), mainly attributed to their versatile membrane properties in biological fluids. Meanwhile, the promising biomedical applications based on nanoplatforms inspired by diverse moieties, such as selective drug delivery in targeted sites and effective vaccine development for disease prevention, have also been outlined. Finally, the potential challenges and future prospects of the biomimetic membrane-cloaked nanoplatforms are also discussed.

Deuterated detergents for structural and functional studies of membrane proteins: Properties, chemical synthesis and applications.

PubMed

Hiruma-Shimizu, Kazumi; Shimizu, Hiroki; Thompson, Gary S; Kalverda, Arnout P; Patching, Simon G

2015-01-01

Detergents are amphiphilic compounds that have crucial roles in the extraction, purification and stabilization of integral membrane proteins and in experimental studies of their structure and function. One technique that is highly dependent on detergents for solubilization of membrane proteins is solution-state NMR spectroscopy, where detergent micelles often serve as the best membrane mimetic for achieving particle sizes that tumble fast enough to produce high-resolution and high-sensitivity spectra, although not necessarily the best mimetic for a biomembrane. For achieving the best quality NMR spectra, detergents with partial or complete deuteration can be used, which eliminate interfering proton signals coming from the detergent itself and also eliminate potential proton relaxation pathways and strong dipole-dipole interactions that contribute line broadening effects. Deuterated detergents have also been used to solubilize membrane proteins for other experimental techniques including small angle neutron scattering and single-crystal neutron diffraction and for studying membrane proteins immobilized on gold electrodes. This is a review of the properties, chemical synthesis and applications of detergents that are currently commercially available and/or that have been synthesized with partial or complete deuteration. Specifically, the detergents are sodium dodecyl sulphate (SDS), lauryldimethylamine-oxide (LDAO), n-octyl-β-D-glucoside (β-OG), n-dodecyl-β-D-maltoside (DDM) and fos-cholines including dodecylphosphocholine (DPC). The review also considers effects of deuteration, detergent screening and guidelines for detergent selection. Although deuterated detergents are relatively expensive and not always commercially available due to challenges associated with their chemical synthesis, they will continue to play important roles in structural and functional studies of membrane proteins, especially using solution-state NMR.

Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

PubMed Central

Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

2011-01-01

The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane

PubMed Central

Richardson, Lynn G. L.; Paila, Yamuna D.; Siman, Steven R.; Chen, Yi; Smith, Matthew D.; Schnell, Danny J.

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus. PMID:24966864

Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

PubMed

Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

2014-01-01

The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

Nanoclustering as a dominant feature of plasma membrane organization.

PubMed

Garcia-Parajo, Maria F; Cambi, Alessandra; Torreno-Pina, Juan A; Thompson, Nancy; Jacobson, Ken

2014-12-01

Early studies have revealed that some mammalian plasma membrane proteins exist in small nanoclusters. The advent of super-resolution microscopy has corroborated and extended this picture, and led to the suggestion that many, if not most, membrane proteins are clustered at the plasma membrane at nanoscale lengths. In this Commentary, we present selected examples of glycosylphosphatidyl-anchored proteins, Ras family members and several immune receptors that provide evidence for nanoclustering. We advocate the view that nanoclustering is an important part of the hierarchical organization of proteins in the plasma membrane. According to this emerging picture, nanoclusters can be organized on the mesoscale to form microdomains that are capable of supporting cell adhesion, pathogen binding and immune cell-cell recognition amongst other functions. Yet, a number of outstanding issues concerning nanoclusters remain open, including the details of their molecular composition, biogenesis, size, stability, function and regulation. Notions about these details are put forth and suggestions are made about nanocluster function and why this general feature of protein nanoclustering appears to be so prevalent. © 2014. Published by The Company of Biologists Ltd.

Tetraspanin-enriched microdomains: a functional unit in cell plasma membranes.

PubMed

Yáñez-Mó, María; Barreiro, Olga; Gordon-Alonso, Mónica; Sala-Valdés, Mónica; Sánchez-Madrid, Francisco

2009-09-01

Membrane lipids and proteins are non-randomly distributed and are unable to diffuse freely in the plane of the membrane. This is because of multiple constraints imposed both by the cortical cytoskeleton and by the preference of lipids and proteins to cluster into diverse and specialized membrane domains, including tetraspanin-enriched microdomains, glycosylphosphatidyl inositol-linked proteins nanodomains and caveolae, among others. Recent biophysical characterization of tetraspanin-enriched microdomains suggests that they might be specially suited for the regulation of avidity of adhesion receptors and the compartmentalization of enzymatic activities. Moreover, modulation by tetraspanins of the function of adhesion receptors involved in inflammation, lymphocyte activation, cancer and pathogen infection suggests potential as therapeutic targets. This review explores this emerging picture of tetraspanin microdomains and discusses the implications for cell adhesion, proteolysis and pathogenesis.

The structure and function of cell membranes studied by atomic force microscopy.

PubMed

Shi, Yan; Cai, Mingjun; Zhou, Lulu; Wang, Hongda

2018-01-01

The cell membrane, involved in almost all communications of cells and surrounding matrix, is one of the most complicated components of cells. Lack of suitable methods for the detection of cell membranes in vivo has sparked debates on the biochemical composition and structure of cell membranes over half a century. The development of single molecule techniques, such as AFM, SMFS, and TREC, provides a versatile platform for imaging and manipulating cell membranes in biological relevant environments. Here, we discuss the latest developments in AFM and the progress made in cell membrane research. In particular, we highlight novel structure models and dynamic processes, including the mechanical properties of the cell membranes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expanded polyglutamine embedded in the endoplasmic reticulum causes membrane distortion and coincides with Bax insertion

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

Ueda, Masashi; Li, Shimo; Itoh, Masanori

The endoplasmic reticulum (ER) is important in various cellular functions, such as secretary and membrane protein biosynthesis, lipid synthesis, and calcium storage. ER stress, including membrane distortion, is associated with many diseases such as Huntington's disease. In particular, nuclear envelope distortion is related to neuronal cell death associated with polyglutamine. However, the mechanism by which polyglutamine causes ER membrane distortion remains unclear. We used electron microscopy, fluorescence protease protection assay, and alkaline treatment to analyze the localization of polyglutamine in cells. We characterized polyglutamine embedded in the ER membrane and noted an effect on morphology, including the dilation of ERmore » luminal space and elongation of ER-mitochondria contact sites, in addition to the distortion of the nuclear envelope. The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. These results demonstrated that the ER membrane may be a target of polyglutamine, which triggers cell death through Bax. -- Highlights: •We characterized polyglutamine embedded in the ER membrane. •The polyglutamine embedded in the ER membrane was observed at the same time as Bax insertion. •The ER membrane may be a target of polyglutamine, which triggers cell death.« less

Functionalized carbon nanotubes for potential medicinal applications.

PubMed

Zhang, Yi; Bai, Yuhong; Yan, Bing

2010-06-01

Functionalized carbon nanotubes display unique properties that enable a variety of medicinal applications, including the diagnosis and treatment of cancer, infectious diseases and central nervous system disorders, and applications in tissue engineering. These potential applications are particularly encouraged by their ability to penetrate biological membranes and relatively low toxicity. High aspect ratio, unique optical property and the likeness as small molecule make carbon nanotubes an unusual allotrope of element carbon. After functionalization, carbon nanotubes display potentials for a variety of medicinal applications, including the diagnosis and treatment of cancer, infectious diseases and central nervous system disorders, and applications in tissue engineering. These potential applications are particularly encouraged by their ability to penetrate biological membranes and relatively low toxicity. (c) 2010 Elsevier Ltd. All rights reserved.

Fabrication and Water Treatment Application of Carbon Nanotubes (CNTs)-Based Composite Membranes: A Review

PubMed Central

Ma, Lining; Dong, Xinfa; Chen, Mingliang; Zhu, Li; Wang, Chaoxian; Yang, Fenglin; Dong, Yingchao

2017-01-01

Membrane separation technology is widely explored for various applications, such as water desalination and wastewater treatment, which can alleviate the global issue of fresh water scarcity. Specifically, carbon nanotubes (CNTs)-based composite membranes are increasingly of interest due to the combined merits of CNTs and membrane separation, offering enhanced membrane properties. This article first briefly discusses fabrication and growth mechanisms, characterization and functionalization techniques of CNTs, and then reviews the fabrication methods for CNTs-based composite membranes in detail. The applications of CNTs-based composite membranes in water treatment are comprehensively reviewed, including seawater or brine desalination, oil-water separation, removal of heavy metal ions and emerging pollutants as well as membrane separation coupled with assistant techniques. Furthermore, the future direction and perspective for CNTs-based composite membranes are also briefly outlined. PMID:28335452

Fabrication and Water Treatment Application of Carbon Nanotubes (CNTs)-Based Composite Membranes: A Review.

PubMed

Ma, Lining; Dong, Xinfa; Chen, Mingliang; Zhu, Li; Wang, Chaoxian; Yang, Fenglin; Dong, Yingchao

2017-03-18

Membrane separation technology is widely explored for various applications, such as water desalination and wastewater treatment, which can alleviate the global issue of fresh water scarcity. Specifically, carbon nanotubes (CNTs)-based composite membranes are increasingly of interest due to the combined merits of CNTs and membrane separation, offering enhanced membrane properties. This article first briefly discusses fabrication and growth mechanisms, characterization and functionalization techniques of CNTs, and then reviews the fabrication methods for CNTs-based composite membranes in detail. The applications of CNTs-based composite membranes in water treatment are comprehensively reviewed, including seawater or brine desalination, oil-water separation, removal of heavy metal ions and emerging pollutants as well as membrane separation coupled with assistant techniques. Furthermore, the future direction and perspective for CNTs-based composite membranes are also briefly outlined.

The big and intricate dreams of little organelles: Embracing complexity in the study of membrane traffic.

PubMed

Liu, Allen P; Botelho, Roberto J; Antonescu, Costin N

2017-09-01

Compartmentalization of eukaryotic cells into dynamic organelles that exchange material through regulated membrane traffic governs virtually every aspect of cellular physiology including signal transduction, metabolism and transcription. Much has been revealed about the molecular mechanisms that control organelle dynamics and membrane traffic and how these processes are regulated by metabolic, physical and chemical cues. From this emerges the understanding of the integration of specific organellar phenomena within complex, multiscale and nonlinear regulatory networks. In this review, we discuss systematic approaches that revealed remarkable insight into the complexity of these phenomena, including the use of proximity-based proteomics, high-throughput imaging, transcriptomics and computational modeling. We discuss how these methods offer insights to further understand molecular versatility and organelle heterogeneity, phenomena that allow a single organelle population to serve a range of physiological functions. We also detail on how transcriptional circuits drive organelle adaptation, such that organelles may shift their function to better serve distinct differentiation and stress conditions. Thus, organelle dynamics and membrane traffic are functionally heterogeneous and adaptable processes that coordinate with higher-order system behavior to optimize cell function under a range of contexts. Obtaining a comprehensive understanding of organellar phenomena will increasingly require combined use of reductionist and system-based approaches. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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.

The interactions of peripheral membrane proteins with biological membranes

DOE PAGES

Johs, Alexander; Whited, A. M.

2015-07-29

The interactions of peripheral proteins with membrane surfaces are critical to many biological processes, including signaling, recognition, membrane trafficking, cell division and cell structure. On a molecular level, peripheral membrane proteins can modulate lipid composition, membrane dynamics and protein-protein interactions. Biochemical and biophysical studies have shown that these interactions are in fact highly complex, dominated by several different types of interactions, and have an interdependent effect on both the protein and membrane. Here we examine three major mechanisms underlying the interactions between peripheral membrane proteins and membranes: electrostatic interactions, hydrophobic interactions, and fatty acid modification of proteins. While experimental approachesmore » continue to provide critical insights into specific interaction mechanisms, emerging bioinformatics resources and tools contribute to a systems-level picture of protein-lipid interactions. Through these recent advances, we begin to understand the pivotal role of protein-lipid interactions underlying complex biological functions at membrane interfaces.« less

Engineering multi-functional bacterial outer membrane vesicles as modular nanodevices for biosensing and bioimaging.

PubMed

Chen, Qi; Rozovsky, Sharon; Chen, Wilfred

2017-07-04

Outer membrane vesicles (OMVs) are proteoliposomes derived from the outer membrane and periplasmic space of many Gram-negative bacteria including E. coli as part of their natural growth cycle. Inspired by the natural ability of E. coli to sort proteins to both the exterior and interior of OMVs, we reported here a one-pot synthesis approach to engineer multi-functionalized OMV-based sensors for both antigen binding and signal generation. SlyB, a native lipoprotein, was used a fusion partner to package nanoluciferase (Nluc) within OMVs, while a previously developed INP-Scaf3 surface scaffold was fused to the Z-domain for antibody recruiting. The multi-functionalized OMVs were used for thrombin detection with a detection limit of 0.5 nM, comparable to other detection methods. Using the cohesin domains inserted between the Z-domain and INP, these engineered OMVs were further functionalized with a dockerin-tagged GFP for cancer cell imaging.

Molecular dynamics simulations of trihalomethanes removal from water using boron nitride nanosheets.

PubMed

Azamat, Jafar; Khataee, Alireza; Joo, Sang Woo

2016-04-01

Molecular dynamics simulations were performed to investigate the separation of trihalomethanes (THMs) from water using boron nitride nanosheets (BNNSs). The studied systems included THM molecules and a functionalized BNNS membrane immersed in an aqueous solution. An external pressure was applied to the z axis of the systems. Two functionalized BNNSs with large fluorinated-hydrogenated pore (F-H-pores) and small hydrogen-hydroxyl pore (H-OH-pores) were used. The pores of the BNNS membrane were obtained by passivating each nitrogen and boron atoms at the pore edges with fluorine and hydrogen atoms in the large pore or with hydroxyl and hydrogen atoms in the small pore. The results show that the BNNS with a small functionalized pore was impermeable to THM molecules, in contrast to the BNNS with a large functionalized pore. Using these membranes, water contaminants can be removed at lower cost.

GSL-enriched membrane microdomains in innate immune responses.

PubMed

Nakayama, Hitoshi; Ogawa, Hideoki; Takamori, Kenji; Iwabuchi, Kazuhisa

2013-06-01

Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host-pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.

OXPHOS-Dependent Cells Identify Environmental Disruptors of Mitochondrial Function

EPA Science Inventory

Mitochondrial dysfunction is associated with numerous chronic diseases including metabolic syndrome. Environmental chemicals can impair mitochondrial function through numerous mechanisms such as membrane disruption, complex inhibition and electron transport chain uncoupling. Curr...

Specificity of Intramembrane Protein–Lipid Interactions

PubMed Central

Contreras, Francesc-Xabier; Ernst, Andreas Max; Wieland, Felix; Brügger, Britta

2011-01-01

Our concept of biological membranes has markedly changed, from the fluid mosaic model to the current model that lipids and proteins have the ability to separate into microdomains, differing in their protein and lipid compositions. Since the breakthrough in crystallizing membrane proteins, the most powerful method to define lipid-binding sites on proteins has been X-ray and electron crystallography. More recently, chemical biology approaches have been developed to analyze protein–lipid interactions. Such methods have the advantage of providing highly specific cellular probes. With the advent of novel tools to study functions of individual lipid species in membranes together with structural analysis and simulations at the atomistic resolution, a growing number of specific protein–lipid complexes are defined and their functions explored. In the present article, we discuss the various modes of intramembrane protein–lipid interactions in cellular membranes, including examples for both annular and nonannular bound lipids. Furthermore, we will discuss possible functional roles of such specific protein–lipid interactions as well as roles of lipids as chaperones in protein folding and transport. PMID:21536707

Comparison of the Impact of High-Flux Dialysis on Mortality in Hemodialysis Patients with and without Residual Renal Function

PubMed Central

Kim, Hyung Wook; Kim, Su-Hyun; Kim, Young Ok; Jin, Dong Chan; Song, Ho Chul; Choi, Euy Jin; Kim, Yong-Lim; Kim, Yon-Su; Kang, Shin-Wook; Kim, Nam-Ho; Yang, Chul Woo; Kim, Yong Kyun

2014-01-01

Background The effect of flux membranes on mortality in hemodialysis (HD) patients is controversial. Residual renal function (RRF) has shown to not only be as a predictor of mortality but also a contributor to β2-microglobulin clearance in HD patients. Our study aimed to determine the interaction of residual renal function with dialyzer membrane flux on mortality in HD patients. Methods HD Patients were included from the Clinical Research Center registry for End Stage Renal Disease, a prospective observational cohort study in Korea. Cox proportional hazards regression models were used to study the association between use of high-flux dialysis membranes and all-cause mortality with RRF and without RRF. The primary outcome was all-cause mortality. Results This study included 893 patients with 24 h-residual urine volume ≥100 ml (569 and 324 dialyzed using low-flux and high-flux dialysis membranes, respectively) and 913 patients with 24 h-residual urine volume <100 ml (570 and 343 dialyzed using low-flux and high-flux dialysis membranes, respectively). After a median follow-up period of 31 months, mortality was not significantly different between the high and low-flux groups in patients with 24 h-residual urine volume ≥100 ml (HR 0.86, 95% CI, 0.38–1.95, P = 0.723). In patients with 24 h-residual urine volume <100 ml, HD using high-flux dialysis membrane was associated with decreased mortality compared to HD using low-flux dialysis membrane in multivariate analysis (HR 0.40, 95% CI, 0.21–0.78, P = 0.007). Conclusions Our data showed that HD using high-flux dialysis membranes had a survival benefit in patients with 24 h-residual urine volume <100 ml, but not in patients with 24 h-residual urine volume ≥100 ml. These findings suggest that high-flux dialysis rather than low-flux dialysis might be considered in HD patients without RRF. PMID:24906205

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.

A high stability Ni-La0.5Ce0.5O2-δ asymmetrical metal-ceramic membrane for hydrogen separation and generation

NASA Astrophysics Data System (ADS)

Zhu, Zhiwen; Sun, Wenping; Wang, Zhongtao; Cao, Jiafeng; Dong, Yingchao; Liu, Wei

2015-05-01

In this work, hydrogen permeation properties of Ni-La0.5Ce0.5O2-δ (LDC) asymmetrical cermet membrane are investigated, including hydrogen fluxes (JH2) under different hydrogen partial pressures, the influence of water vapor on JH2 and the long-term stability of the membrane operating under the containing-CO2 atmosphere. Ni-LDC asymmetrical membrane shows the best hydrogen permeability among LDC-based hydrogen separation membranes, inferior to Ni-BaZr0.1Ce0.7Y0.2O3-δ asymmetrical membrane. The water vapor in feed gas is beneficial to hydrogen transport process, which promote an increase of JH2 from 5.64 × 10-8 to 6.83 × 10-8 mol cm-2 s-1 at 900 °C. Stability testing of hydrogen permeation suggests that Ni-LDC membrane remains stable against CO2. A dual function of combining hydrogen separation and generation can be realized by humidifying the sweep gas and enhance the hydrogen output by 1.0-1.5 times. Ni-LDC membrane exhibits desirable performance and durability in dual-function mode. Morphologies and phase structures of the membrane after tests are also characterized by SEM and XRD.

Not all transmembrane helices are born equal: Towards the extension of the sequence homology concept to membrane proteins

PubMed Central

2011-01-01

Background Sequence homology considerations widely used to transfer functional annotation to uncharacterized protein sequences require special precautions in the case of non-globular sequence segments including membrane-spanning stretches composed of non-polar residues. Simple, quantitative criteria are desirable for identifying transmembrane helices (TMs) that must be included into or should be excluded from start sequence segments in similarity searches aimed at finding distant homologues. Results We found that there are two types of TMs in membrane-associated proteins. On the one hand, there are so-called simple TMs with elevated hydrophobicity, low sequence complexity and extraordinary enrichment in long aliphatic residues. They merely serve as membrane-anchoring device. In contrast, so-called complex TMs have lower hydrophobicity, higher sequence complexity and some functional residues. These TMs have additional roles besides membrane anchoring such as intra-membrane complex formation, ligand binding or a catalytic role. Simple and complex TMs can occur both in single- and multi-membrane-spanning proteins essentially in any type of topology. Whereas simple TMs have the potential to confuse searches for sequence homologues and to generate unrelated hits with seemingly convincing statistical significance, complex TMs contain essential evolutionary information. Conclusion For extending the homology concept onto membrane proteins, we provide a necessary quantitative criterion to distinguish simple TMs (and a sufficient criterion for complex TMs) in query sequences prior to their usage in homology searches based on assessment of hydrophobicity and sequence complexity of the TM sequence segments. Reviewers This article was reviewed by Shamil Sunyaev, L. Aravind and Arcady Mushegian. PMID:22024092

Non-covalent bonding interaction of surfactants with functionalized carbon nanotubes in proton exchange membranes for fuel cell applications.

PubMed

Sayeed, M Abu; Kim, Young Ho; Park, Younjin; Gopalan, A I; Lee, Kwang-Pill; Choi, Sang-June

2013-11-01

Dispersion of functionalized multiwalled carbon nanotubes (MWCNTs) in proton exchange membranes (PEMs) was conducted via non-covalent bonding between benzene rings of various surfactants and functionalized MWCNTs. In the solution casting method, dispersion of functionalized MWCNTs in PEMs such as Nafion membranes is a critical issue. In this study, 1 wt.% pristine MWCNTs (p-MWCNTs) and oxidized MWCNTs (ox-MWCNTs) were reinforced in Nafion membranes by adding 0.1-0.5 wt.% of a surfactant such as benzalkonium chloride (BKC) as a cationic surfactant with a benzene ring, Tween-80 as a nonanionic surfactant without a benzene ring, sodium dodecylsulfonate (SDS) as an anionic surfactant without a benzene ring, or sodium dodecylben-zenesulfonate (SDBS) as an anionic surfactant with a benzene ring and their effects on the dispersion of nanocomposites were then observed. Among these surfactants, those with benzene rings such as BKC and SDBS produced enhanced dispersion via non-covalent bonding interaction between CNTs and surfactants. Specifically, the surfactants were adsorbed onto the surface of functionalized MWCNTs, where they prevented re-aggregation of MWCNTs in the nanocomposites. Furthermore, the prepared CNTs reinforced nanocomposite membranes showed reduced methanol uptake values while the ion exchange capacity values were maintained. The enhanced properties, including thermal property of the CNTs reinforced PEMs with surfactants, could be applicable to fuel cell applications.

On the mechanics of growing thin biological membranes

NASA Astrophysics Data System (ADS)

Rausch, Manuel K.; Kuhl, Ellen

2014-02-01

Despite their seemingly delicate appearance, thin biological membranes fulfill various crucial roles in the human body and can sustain substantial mechanical loads. Unlike engineering structures, biological membranes are able to grow and adapt to changes in their mechanical environment. Finite element modeling of biological growth holds the potential to better understand the interplay of membrane form and function and to reliably predict the effects of disease or medical intervention. However, standard continuum elements typically fail to represent thin biological membranes efficiently, accurately, and robustly. Moreover, continuum models are typically cumbersome to generate from surface-based medical imaging data. Here we propose a computational model for finite membrane growth using a classical midsurface representation compatible with standard shell elements. By assuming elastic incompressibility and membrane-only growth, the model a priori satisfies the zero-normal stress condition. To demonstrate its modular nature, we implement the membrane growth model into the general-purpose non-linear finite element package Abaqus/Standard using the concept of user subroutines. To probe efficiently and robustness, we simulate selected benchmark examples of growing biological membranes under different loading conditions. To demonstrate the clinical potential, we simulate the functional adaptation of a heart valve leaflet in ischemic cardiomyopathy. We believe that our novel approach will be widely applicable to simulate the adaptive chronic growth of thin biological structures including skin membranes, mucous membranes, fetal membranes, tympanic membranes, corneoscleral membranes, and heart valve membranes. Ultimately, our model can be used to identify diseased states, predict disease evolution, and guide the design of interventional or pharmaceutic therapies to arrest or revert disease progression.

On the mechanics of growing thin biological membranes

PubMed Central

Rausch, Manuel K.; Kuhl, Ellen

2013-01-01

Despite their seemingly delicate appearance, thin biological membranes fulfill various crucial roles in the human body and can sustain substantial mechanical loads. Unlike engineering structures, biological membranes are able to grow and adapt to changes in their mechanical environment. Finite element modeling of biological growth holds the potential to better understand the interplay of membrane form and function and to reliably predict the effects of disease or medical intervention. However, standard continuum elements typically fail to represent thin biological membranes efficiently, accurately, and robustly. Moreover, continuum models are typically cumbersome to generate from surface-based medical imaging data. Here we propose a computational model for finite membrane growth using a classical midsurface representation compatible with standard shell elements. By assuming elastic incompressibility and membrane-only growth, the model a priori satisfies the zero-normal stress condition. To demonstrate its modular nature, we implement the membrane growth model into the general-purpose non-linear finite element package Abaqus/Standard using the concept of user subroutines. To probe efficiently and robustness, we simulate selected benchmark examples of growing biological membranes under different loading conditions. To demonstrate the clinical potential, we simulate the functional adaptation of a heart valve leaflet in ischemic cardiomyopathy. We believe that our novel approach will be widely applicable to simulate the adaptive chronic growth of thin biological structures including skin membranes, mucous membranes, fetal membranes, tympanic membranes, corneoscleral membranes, and heart valve membranes. Ultimately, our model can be used to identify diseased states, predict disease evolution, and guide the design of interventional or pharmaceutic therapies to arrest or revert disease progression. PMID:24563551

Involvement of glycosphingolipid-enriched lipid rafts in inflammatory responses.

PubMed

Iwabuchi, Kazuhisa

2015-01-01

Glycosphingolipids (GSLs) are membrane components consisting of hydrophobic ceramide and hydrophilic sugar moieties. GSLs cluster with cholesterol in cell membranes to form GSL-enriched lipid rafts. Biochemical analyses have demonstrated that GSL-enriched lipid rafts contain several kinds of transducer molecules, including Src family kinases. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms, is highly expressed on the plasma membranes of human phagocytes, and forms lipid rafts containing the Src family tyrosine kinase Lyn. LacCer-enriched lipid rafts mediate immunological and inflammatory reactions, including superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. LacCer also serves as a signal transduction molecule for functions mediated by CD11b/CD18-integrin (αM/β2-integrin, CR3, Mac-1), as well as being associated with several key cellular processes. LacCer recruits PCKα/ε and phospholipase A2 to stimulate PECAM-1 expression in human monocytes and their adhesion to endothelial cells, as well as regulating β1-integrin clustering and endocytosis on cell surfaces. This review describes the organizational and inflammation-related functions of LacCer-enriched lipid rafts.

Mechanisms Underlying the Essential Role of Mitochondrial Membrane Lipids in Yeast Chronological Aging

PubMed Central

Medkour, Younes; Dakik, Paméla; McAuley, Mélissa; Mohammad, Karamat; Mitrofanova, Darya

2017-01-01

The functional state of mitochondria is vital to cellular and organismal aging in eukaryotes across phyla. Studies in the yeast Saccharomyces cerevisiae have provided evidence that age-related changes in some aspects of mitochondrial functionality can create certain molecular signals. These signals can then define the rate of cellular aging by altering unidirectional and bidirectional communications between mitochondria and other organelles. Several aspects of mitochondrial functionality are known to impact the replicative and/or chronological modes of yeast aging. They include mitochondrial electron transport, membrane potential, reactive oxygen species, and protein synthesis and proteostasis, as well as mitochondrial synthesis of iron-sulfur clusters, amino acids, and NADPH. Our recent findings have revealed that the composition of mitochondrial membrane lipids is one of the key aspects of mitochondrial functionality affecting yeast chronological aging. We demonstrated that exogenously added lithocholic bile acid can delay chronological aging in yeast because it elicits specific changes in mitochondrial membrane lipids. These changes allow mitochondria to operate as signaling platforms that delay yeast chronological aging by orchestrating an institution and maintenance of a distinct cellular pattern. In this review, we discuss molecular and cellular mechanisms underlying the essential role of mitochondrial membrane lipids in yeast chronological aging. PMID:28593023

Structure-function analysis of HKE4, a member of the new LIV-1 subfamily of zinc transporters.

PubMed Central

Taylor, Kathryn M; Morgan, Helen E; Johnson, Andrea; Nicholson, Robert I

2004-01-01

The KE4 proteins are an emerging group of proteins with little known functional data. In the present study, we report the first characterization of the recombinant human KE4 protein in mammalian cells. The KE4 sequences are included in the subfamily of ZIP (Zrt-, Irt-like Proteins) zinc transporters, which we have termed LZT (LIV-1 subfamily of ZIP zinc Transporters). All these LZT sequences contain similarities to ZIP transporters, including the consensus sequence in transmembrane domain IV, which is essential for zinc transport. However, the new LZT subfamily can be separated from other ZIP transporters by the presence of a highly conserved potential metalloprotease motif (HEXPHEXGD) in transmembrane domain V. Here we report the location of HKE4 on intracellular membranes, including the endoplasmic reticulum, and its ability to increase the intracellular free zinc as measured with the zinc-specific fluorescent dye, Newport Green, in a time-, temperature- and concentration-dependent manner. This is in contrast with the zinc influx ability of another LZT protein, LIV-1, which was due to its plasma membrane location. Therefore we have added to the functionality of LZT proteins by reporting their ability to increase intracellular-free zinc, whether they are located on the plasma membrane or on intracellular membranes. This result, in combination with the crucial role that zinc plays in cell growth, emphasizes the importance of this new LZT subfamily, including the KE4 sequences, in the control of intracellular zinc homoeostasis, aberrations of which can lead to diseases such as cancer, immunological disorders and neurological dysfunction. PMID:14525538

Common α2A and α2C adrenergic receptor polymorphisms do not affect plasma membrane trafficking.

PubMed

Hurt, Carl M; Sorensen, Matt W; Angelotti, Timothy

2014-06-01

Various naturally occurring polymorphic forms of human G protein-coupled receptors (GPCRs) have been identified and linked to diverse pathological diseases, including receptors for vasopressin type 2 (nephrogenic diabetes insipidus) and gonadotropin releasing hormone (hypogonadotropic hypogonadism). In most cases, polymorphic amino acid mutations disrupt protein folding, altering receptor function as well as plasma membrane expression. Other pathological GPCR variants have been found that do not alter receptor function, but instead affect only plasma membrane trafficking (e.g., delta opiate and histamine type 1 receptors). Thus, altered membrane trafficking with retained receptor function may be another mechanism causing polymorphic GPCR dysfunction. Two common human α2A and α2C adrenergic receptor (AR) variants have been identified (α2A N251K and α2C Δ322-325 ARs), but pharmacological analysis of ligand binding and second messenger signaling has not consistently demonstrated altered receptor function. However, possible alterations in plasma membrane trafficking have not been investigated. We utilized a systematic approach previously developed for the study of GPCR trafficking motifs and accessory proteins to assess whether these α2 AR variants affected intracellular trafficking or plasma membrane expression. By combining immunofluorescent microscopy, glycosidic processing analysis, and quantitative fluorescent-activated cell sorting (FACS), we demonstrate that neither variant receptor had altered intracellular localization, glycosylation, nor plasma membrane expression compared to wild-type α2 ARs. Therefore, pathopharmacological properties of α2A N251K and α2C Δ322-325 ARs do not appear to be due to altered receptor pharmacology or plasma membrane trafficking, but may involve interactions with other intracellular signaling cascades or proteins.

Devices for overcoming biological barriers: the use of physical forces to disrupt the barriers.

PubMed

Mitragotri, Samir

2013-01-01

Overcoming biological barriers including skin, mucosal membranes, blood brain barrier as well as cell and nuclear membrane constitutes a key hurdle in the field of drug delivery. While these barriers serve the natural protective function in the body, they limit delivery of drugs into the body. A variety of methods have been developed to overcome these barriers including formulations, targeting peptides and device-based technologies. This review focuses on the use of physical methods including acoustic devices, electric devices, high-pressure devices, microneedles and optical devices for disrupting various barriers in the body including skin and other membranes. A summary of the working principles of these devices and their ability to enhance drug delivery is presented. Copyright © 2012. Published by Elsevier B.V.

Building membrane nanopores

NASA Astrophysics Data System (ADS)

Howorka, Stefan

2017-07-01

Membrane nanopores--hollow nanoscale barrels that puncture biological or synthetic membranes--have become powerful tools in chemical- and biosensing, and have achieved notable success in portable DNA sequencing. The pores can be self-assembled from a variety of materials, including proteins, peptides, synthetic organic compounds and, more recently, DNA. But which building material is best for which application, and what is the relationship between pore structure and function? In this Review, I critically compare the characteristics of the different building materials, and explore the influence of the building material on pore structure, dynamics and function. I also discuss the future challenges of developing nanopore technology, and consider what the next-generation of nanopore structures could be and where further practical applications might emerge.

Conserved features in TamA enable interaction with TamB to drive the activity of the translocation and assembly module

PubMed Central

Selkrig, Joel; Belousoff, Matthew J.; Headey, Stephen J.; Heinz, Eva; Shiota, Takuya; Shen, Hsin-Hui; Beckham, Simone A.; Bamert, Rebecca S.; Phan, Minh-Duy; Schembri, Mark A.; Wilce, Matthew C.J.; Scanlon, Martin J.; Strugnell, Richard A.; Lithgow, Trevor

2015-01-01

The biogenesis of membranes from constituent proteins and lipids is a fundamental aspect of cell biology. In the case of proteins assembled into bacterial outer membranes, an overarching question concerns how the energy required for protein insertion and folding is accessed at this remote location of the cell. The translocation and assembly module (TAM) is a nanomachine that functions in outer membrane biogenesis and virulence in diverse bacterial pathogens. Here we demonstrate the interactions through which TamA and TamB subunits dock to bridge the periplasm, and unite the outer membrane aspects to the inner membrane of the bacterial cell. We show that specific functional features in TamA have been conserved through evolution, including residues surrounding the lateral gate and an extensive surface of the POTRA domains. Analysis by nuclear magnetic resonance spectroscopy and small angle X-ray scattering document the characteristic structural features of these POTRA domains and demonstrate rigidity in solution. Quartz crystal microbalance measurements pinpoint which POTRA domain specifically docks the TamB subunit of the nanomachine. We speculate that the POTRA domain of TamA functions as a lever arm in order to drive the activity of the TAM, assembling proteins into bacterial outer membranes. PMID:26243377

Seasonal changes in minor membrane phospholipid classes, sterols and tocopherols in overwintering insect, Pyrrhocoris apterus.

PubMed

Koštál, Vladimír; Urban, Tomáš; Rimnáčová, Lucie; Berková, Petra; Simek, Petr

2013-09-01

Ectotherm animals including insects are known to undergo seasonal restructuring of the cell membranes in order to keep their functionality and/or protect their structural integrity at low body temperatures. Studies on insects so far focused either on fatty acids or on composition of molecular species in major phospholipid classes. Here we extend the scope of analysis and bring results on seasonal changes in minor phospholipid classes, lysophospholipids (LPLs), free fatty acids, phytosterols and tocopherols in heteropteran insect, Pyrrhocoris apterus. We found that muscle tissue contains unusually high amounts of LPLs. Muscle and fat body tissues also contain high amounts of β-sitosterol and campesterol, two phytosterols derived from plant food, while only small amounts of cholesterol are present. In addition, two isomers (γ and δ) of tocopherol (vitamin E) are present in quantities comparable to, or even higher than phytosterols in both tissues. Distinct seasonal patterns of sterol and tocopherol concentrations were observed showing a minimum in reproductively active bugs in summer and a maximum in diapausing, cold-acclimated bugs in winter. Possible adaptive meanings of such changes are discussed including: preventing the unregulated transition of membrane lipids from functional liquid crystalline phase to non-functional gel phase; decreasing the rates of ion/solute leakage; silencing the activities of membrane bound enzymes and receptors; and counteracting the higher risk of oxidative damage to PUFA in winter membranes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Grafted functional groups on expanded tetrafluoroethylene (ePTFE) support for fuel cell and water transport membranes

DOEpatents

Fuller, Timothy J.; Jiang, Ruichun

2017-01-24

A method for forming a modified solid polymer includes a step of contacting a solid fluorinated polymer with a sodium sodium-naphthalenide solution to form a treated fluorinated solid polymer. The treated fluorinated solid polymer is contacted with carbon dioxide, sulfur dioxide, or sulfur trioxide to form a solid grafted fluorinated polymer. Characteristically, the grafted fluorinated polymer includes appended CO.sub.2H or SO.sub.2H or SO.sub.3H groups. The solid grafted fluorinated polymer is advantageously incorporated into a fuel cell as part of the ion-conducting membrane or a water transport membrane in a humidifier.

Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells.

PubMed

Andoh, Yoshimichi; Okazaki, Susumu; Ueoka, Ryuichi

2013-04-01

Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal-isobaric conditions (310.15K and 1 atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes. Copyright © 2013 Elsevier B.V. All rights reserved.

Functional, Responsive Materials Assembled from Recombinant Oleosin

NASA Astrophysics Data System (ADS)

Hammer, Daniel

Biological cells are surrounded by a plasma membrane made primarily of phospholipids that form a bilayer. This membrane is permselective and compartmentalizes the cell. A simple form of artificial cell is the vesicle, in which a phospholipid bilayer membrane surrounds an aqueous solution. However, there is no a priori reason why a membrane needs to be made of phospholipids. It could be made of any surfactant that forms a bilayer. We have assembled membranes and other structures from the recombinant plant protein oleosin. The ability to assemble from a recombinant protein means that every molecule is identical, we have complete control over the sequence, and hence can build in designer functionality with high fidelity, including adhesion and enzymatic activity. Such incorporation is trivial using the tools of molecular biology. We find that while many variants of oleosin make membranes, others make micelles and sheets. We show how the type of supramolecular structure can be altered by the conditions of solvent, such as ionic strength, and the architecture of the surfactant itself. We show that protease cleavable domains can be incorporated within oleosin, and be engineered to protect other functional domains such as adhesive motifs, to make responsive materials whose activity and shape depend on the action of proteases. We will also present the idea of making ``Franken''-oleosins, where large domains of native oleosin are replaced with domains from other functional proteins, to make hybrids conferred by the donor protein. Thus, we can view oleosin as a template upon which a vast array of designer functionalities can be imparted..

Endoplasmic Reticulum-Plasma Membrane Contact Sites.

PubMed

Saheki, Yasunori; De Camilli, Pietro

2017-06-20

The endoplasmic reticulum (ER) has a broad localization throughout the cell and forms direct physical contacts with all other classes of membranous organelles, including the plasma membrane (PM). A number of protein tethers that mediate these contacts have been identified, and study of these protein tethers has revealed a multiplicity of roles in cell physiology, including regulation of intracellular Ca 2+ dynamics and signaling as well as control of lipid traffic and homeostasis. In this review, we discuss the cross talk between the ER and the PM mediated by direct contacts. We review factors that tether the two membranes, their properties, and their dynamics in response to the functional state of the cell. We focus in particular on the role of ER-PM contacts in nonvesicular lipid transport between the two bilayers mediated by lipid transfer proteins.

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

PubMed

Schuberth, Christian; Wedlich-Söldner, Roland

2015-04-01

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

Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins.

PubMed

Stojanovski, Diana; Guiard, Bernard; Kozjak-Pavlovic, Vera; Pfanner, Nikolaus; Meisinger, Chris

2007-12-03

The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the beta-barrel-specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a beta-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane alpha-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of alpha-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to beta-barrel proteins but also includes the majority of alpha-helical Tom proteins.

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

PubMed

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

2014-02-01

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

Hairpin Folding of HIV gp41 Abrogates Lipid Mixing Function at Physiologic pH and Inhibits Lipid Mixing by Exposed gp41 Constructs†

PubMed Central

Sackett, Kelly; Nethercott, Matthew J.; Shai, Yechiel; Weliky, David P.

2009-01-01

Conformational changes in the HIV gp41 protein are directly correlated with fusion between the HIV and target cell plasma membranes which is the initial step of infection. Key gp41 fusion conformations include an early extended conformation termed pre-hairpin which contains exposed regions and a final low energy conformation termed hairpin which has compact six-helix bundle structure. Current fusion models debate the roles of hairpin and pre-hairpin conformations in the process of membrane merger. In the present work, gp41 constructs have been engineered which correspond to fusion relevant parts of both pre-hairpin and hairpin conformations, and have been analyzed for their ability to induce lipid mixing between membrane vesicles. The data correlate membrane fusion function with the pre-hairpin conformation and suggest that one of the roles of the final hairpin conformation is sequestration of membrane perturbing gp41 regions with consequent loss of the membrane disruption induced earlier by the pre-hairpin structure. To our knowledge, this is the first biophysical study to delineate the membrane fusion potential of gp41 constructs modeling key fusion conformations. PMID:19222185

Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling

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

Brown, Colin D.A.; Sayer, Rachel; Windass, Amy S.

2008-12-15

The aim of this study was to determine whether primary human tubular cell monolayers could provide a powerful tool with which to investigate the renal proximal tubular handling of xenobiotics. Human proximal and distal tubule/collecting duct cells were grown as monolayers on permeable filter supports. After 10 days in culture, proximal tubule cells remained differentiated and expressed a wide palette of transporters at the mRNA level including NaPi-IIa, SGLT1, SGLT2, OCT2, OCTN2, OAT1, OAT3, OAT4, MDR1, MRP2 and BCRP. At the protein level, the expression of a subset of transporters including NaPi-IIa, OAT1 and OAT3 was demonstrated using immunohistochemistry. Analysismore » of the expression of the ATP binding cassette efflux pumps MDR1, MRP2 and BCRP confirmed their apical membrane localisation. At the functional level, tubule cell monolayers retain the necessary machinery to mediate the net secretion of the prototypic substrates; PAH and creatinine. PAH secretion across the monolayer consisted of the uptake of PAH across the basolateral membrane by OAT1 and OAT3 and the apical exit of PAH by a probenecid and MK571-sensitive route consistent with actions of MRP2 or MRP4. Creatinine secretion was by OCT2-mediated uptake at the basolateral membrane and via MDR1 at the apical membrane. Functional expression of MDR1 and BCRP at the apical membrane was also demonstrated using a Hoechst 33342 dye. Similarly, measurement of calcein efflux demonstrated the functional expression of MRP2 at the apical membrane of cell monolayers. In conclusion, human tubular cell monolayers provide a powerful tool to investigate renal xenobiotic handling.« less

Biopores/membrane proteins in synthetic polymer membranes.

PubMed

Garni, Martina; Thamboo, Sagana; Schoenenberger, Cora-Ann; Palivan, Cornelia G

2017-04-01

Mimicking cell membranes by simple models based on the reconstitution of membrane proteins in lipid bilayers represents a straightforward approach to understand biological function of these proteins. This biomimetic strategy has been extended to synthetic membranes that have advantages in terms of chemical and mechanical stability, thus providing more robust hybrid membranes. We present here how membrane proteins and biopores have been inserted both in the membrane of nanosized and microsized compartments, and in planar membranes under various conditions. Such bio-hybrid membranes have new properties (as for example, permeability to ions/molecules), and functionality depending on the specificity of the inserted biomolecules. Interestingly, membrane proteins can be functionally inserted in synthetic membranes provided these have appropriate properties to overcome the high hydrophobic mismatch between the size of the biomolecule and the membrane thickness. Functional insertion of membrane proteins and biopores in synthetic membranes of compartments or in planar membranes is possible by an appropriate selection of the amphiphilic copolymers, and conditions of the self-assembly process. These hybrid membranes have new properties and functionality based on the specificity of the biomolecules and the nature of the synthetic membranes. Bio-hybrid membranes represent new solutions for the development of nanoreactors, artificial organelles or active surfaces/membranes that, by further gaining in complexity and functionality, will promote translational applications. This article is part of a Special Issue entitled: Lipid order/lipid defects and lipid-control of protein activity edited by Dirk Schneider. Copyright © 2016. Published by Elsevier B.V.

Generalized Boussinesq-Scriven surface fluid model with curvature dissipation for liquid surfaces and membranes.

PubMed

Aguilar Gutierrez, Oscar F; Herrera Valencia, Edtson E; Rey, Alejandro D

2017-10-01

Curvature dissipation is relevant in synthetic and biological processes, from fluctuations in semi-flexible polymer solutions, to buckling of liquid columns, tomembrane cell wall functioning. We present a micromechanical model of curvature dissipation relevant to fluid membranes and liquid surfaces based on a parallel surface parameterization and a stress constitutive equation appropriate for anisotropic fluids and fluid membranes.The derived model, aimed at high curvature and high rate of change of curvature in liquid surfaces and membranes, introduces additional viscous modes not included in the widely used 2D Boussinesq-Scriven rheological constitutive equation for surface fluids.The kinematic tensors that emerge from theparallel surface parameterization are the interfacial rate of deformation and the surface co-rotational Zaremba-Jaumann derivative of the curvature, which are used to classify all possibledissipative planar and non-planar modes. The curvature dissipation function that accounts for bending, torsion and twist rates is derived and analyzed under several constraints, including the important inextensional bending mode.A representative application of the curvature dissipation model to the periodic oscillation in nano-wrinkled outer hair cells show how and why curvature dissipation decreases with frequency, and why the 100kHz frequency range is selected. These results contribute to characterize curvature dissipation in membranes and liquid surfaces. Copyright © 2017 Elsevier Inc. All rights reserved.

Amine-functionalized PVA-co-PE nanofibrous membrane as affinity membrane with high adsorption capacity for bilirubin.

PubMed

Wang, Wenwen; Zhang, Hao; Zhang, Zhifeng; Luo, Mengying; Wang, Yuedan; Liu, Qiongzhen; Chen, Yuanli; Li, Mufang; Wang, Dong

2017-02-01

In this study, poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membrane was activated by sodium hydroxide and cyanuric chloride, and then the activated membranes were functionalized by 1,3-propanediamine, hexamethylenediamine and diethylenetriamine to be affinity membranes for bilirubin removal, respectively. The chemical structures and morphologies of membranes were investigated by SEM, FTIR and XPS. And the adsorption ability of different amine-functionalized nanofibrous membranes for bilirubin was characterized. Furthermore, the effects of temperature, initial concentration of bilirubin, NaCl concentration and BSA concentration on the adsorption capacity for bilirubin of diethylenetriamine-functionalized nanofibrous membrane were studied. Results indicated that the adsorption capacity for bilirubin of diethylenetriamine-functionalized nanofibrous membrane could reach 85mg/g membrane when the initial bilirubin concentration was 200mg/L while the adsorption capacity could be increased to 110mg/g membrane if the initial bilirubin concentration was more than 400mg/L. The dynamic adsorption of diethylenetriamine-functionalized nanofibrous membrane showed that the ligands of amine groups on the membrane surface could be used as far as possible by recirculating the plasma with certain flow rates. Therefore, the diethylenetriamine-functionalized PVA-co-PE nanofibrous membrane possessed high adsorption capacity for bilirubin and it can be candidate as affinity membrane for bilirubin removal. Copyright © 2016. Published by Elsevier B.V.

The relevance of polymeric synthetic membranes in topical formulation assessment and drug diffusion study.

PubMed

Ng, Shiow-Fern; Rouse, Jennifer J; Sanderson, Francis D; Eccleston, Gillian M

2012-03-01

Synthetic membranes are composed of thin sheets of polymeric macromolecules that can control the passage of components through them. Generally, synthetic membranes used in drug diffusion studies have one of two functions: skin simulation or quality control. Synthetic membranes for skin simulation, such as the silicone-based membranes polydimethylsiloxane and Carbosil, are generally hydrophobic and rate limiting, imitating the stratum corneum. In contrast, synthetic membranes for quality control, such as cellulose esters and polysulfone, are required to act as a support rather than a barrier. These synthetic membranes also often contain pores; hence, they are called porous membranes. The significance of Franz diffusion studies and synthetic membranes in quality control studies involves an understanding of the fundamentals of synthetic membranes. This article provides a general overview of synthetic membranes, including a brief background of the history and the common applications of synthetic membranes. This review then explores the types of synthetic membranes, the transport mechanisms across them, and their relevance in choosing a synthetic membrane in Franz diffusion cell studies for formulation assessment purposes.

Improved In Vitro and In Vivo Biocompatibility of Graphene Oxide through Surface Modification: Poly(Acrylic Acid)-Functionalization is Superior to PEGylation.

PubMed

Xu, Ming; Zhu, Jianqiang; Wang, Fanfan; Xiong, Yunjing; Wu, Yakun; Wang, Qiuquan; Weng, Jian; Zhang, Zhihong; Chen, Wei; Liu, Sijin

2016-03-22

The unique physicochemical properties of two-dimensional (2D) graphene oxide (GO) could greatly benefit the biomedical field; however, recent research demonstrated that GO could induce in vitro and in vivo toxicity. We determined the mechanism of GO induced toxicity, and our in vitro experiments revealed that pristine GO could impair cell membrane integrity and functions including regulation of membrane- and cytoskeleton-associated genes, membrane permeability, fluidity and ion channels. Furthermore, GO induced platelet depletion, pro-inflammatory response and pathological changes of lung and liver in mice. To improve the biocompatibility of pristine GO, we prepared a series of GO derivatives including aminated GO (GO-NH2), poly(acrylamide)-functionalized GO (GO-PAM), poly(acrylic acid)-functionalized GO (GO-PAA) and poly(ethylene glycol)-functionalized GO (GO-PEG), and compared their toxicity with pristine GO in vitro and in vivo. Among these GO derivatives, GO-PEG and GO-PAA induced less toxicity than pristine GO, and GO-PAA was the most biocompatible one in vitro and in vivo. The differences in biocompatibility were due to the differential compositions of protein corona, especially immunoglobulin G (IgG), formed on their surfaces that determine their cell membrane interaction and cellular uptake, the extent of platelet depletion in blood, thrombus formation under short-term exposure and the pro-inflammatory effects under long-term exposure. Overall, our combined data delineated the key molecular mechanisms underlying the in vivo and in vitro biological behaviors and toxicity of pristine GO, and identified a safer GO derivative that could be used for future applications.

Functional membranes. Present and future

NASA Technical Reports Server (NTRS)

Kunitake, T.

1982-01-01

The present situation and the future development of the functional membrane are discussed. It is expected that functional membranes will play increasingly greater roles in the chemical industry of the coming decade. These membranes are formed from polymer films, liquid membranes or bilayer membranes. The two most important technologies based on the polymeric membrane are reverse osmosis and ion exchange. The liquid membrane is used for separation of ionic species; an extension of the solvent extraction process. By using appropriate ligands and ionophores, highly selective separations are realized. The active transport is made possible if the physical and chemical potentials are applied to the transport process. More advanced functional membranes may be designed on the basis of the synthetic bilayer membrane.

Future directions of electron crystallography.

PubMed

Fujiyoshi, Yoshinori

2013-01-01

In biological science, there are still many interesting and fundamental yet difficult questions, such as those in neuroscience, remaining to be answered. Structural and functional studies of membrane proteins, which are key molecules of signal transduction in neural and other cells, are essential for understanding the molecular mechanisms of many fundamental biological processes. Technological and instrumental advancements of electron microscopy have facilitated comprehension of structural studies of biological components, such as membrane proteins. While X-ray crystallography has been the main method of structure analysis of proteins including membrane proteins, electron crystallography is now an established technique to analyze structures of membrane proteins in the lipid bilayer, which is close to their natural biological environment. By utilizing cryo-electron microscopes with helium-cooled specimen stages, structures of membrane proteins were analyzed at a resolution better than 3 Å. Such high-resolution structural analysis of membrane proteins by electron crystallography opens up the new research field of structural physiology. Considering the fact that the structures of integral membrane proteins in their native membrane environment without artifacts from crystal contacts are critical in understanding their physiological functions, electron crystallography will continue to be an important technology for structural analysis. In this chapter, I will present several examples to highlight important advantages and to suggest future directions of this technique.

A high content in lipid-modified peripheral proteins and integral receptor kinases features in the arabidopsis plasma membrane proteome.

PubMed

Marmagne, Anne; Ferro, Myriam; Meinnel, Thierry; Bruley, Christophe; Kuhn, Lauriane; Garin, Jérome; Barbier-Brygoo, Hélène; Ephritikhine, Geneviève

2007-11-01

The proteomics of plasma membrane has brought to date only scarce and partial information on the actual protein repertoire. In this work, the plant plasma membrane proteome of Arabidopsis thaliana was investigated. A highly purified plasma membrane fraction was washed by NaCl and Na2CO3 salts, and the insoluble fractions were further analyzed by nano-LC-MS/MS. With 446 proteins identified, we hereby describe the largest plasma membrane proteome diversity reported so far. Half of the proteins were predicted to display transmembrane domains and/or to be anchored to the membrane, validating a posteriori the pertinence of the approach. A fine analysis highlighted two main specific and novel features. First, the main functional category is represented by a majority of as yet unreported signaling proteins, including 11% receptor-like kinases. Second, 16% of the identified proteins are predicted to be lipid-modified, specifically involving double lipid linkage through N-terminal myristoylation, S-palmitoylation, C-terminal prenylation, or glycosylphosphatidylinositol anchors. Thus, our approach led for the first time to the identification of a large number of peripheral proteins as part of the plasma membrane and allowed the functionality of the plasma membrane in the cell context to be reconsidered.

Analysis of Membrane Protein Topology in the Plant Secretory Pathway.

PubMed

Guo, Jinya; Miao, Yansong; Cai, Yi

2017-01-01

Topology of membrane proteins provides important information for the understanding of protein function and intermolecular associations. Integrate membrane proteins are generally transported from endoplasmic reticulum (ER) to Golgi and downstream compartments in the plant secretory pathway. Here, we describe a simple method to study membrane protein topology along the plant secretory pathway by transiently coexpressing a fluorescent protein (XFP)-tagged membrane protein and an ER export inhibitor protein, ARF1 (T31N), in tobacco BY-2 protoplast. By fractionation, microsome isolation, and trypsin digestion, membrane protein topology could be easily detected by either direct confocal microscopy imaging or western-blot analysis using specific XFP antibodies. A similar strategy in determining membrane protein topology could be widely adopted and applied to protein analysis in a broad range of eukaryotic systems, including yeast cells and mammalian cells.

Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

NASA Astrophysics Data System (ADS)

Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

2016-04-01

Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner. Electronic supplementary information (ESI) available: Two videos of the experiment are shown in Fig. 5, demonstrating the distinctive characteristics of the peptide pair in a mixed population of cells are available in online. Video S1 shows the experiment in the bright field channel including the green channel (calcein-AM stained unfunctionalized cells) and orange channel (rhodamine labeled liposomes). Video S2 shows the exact same frames but combining the fluorescent channels only, including the blue channel for Hoechst nuclear staining. Both videos consist of 31 frames at a frame rate of 5 fps. The labeled liposomes are injected after frame 1. The videos span a total timeframe of 15 minutes. See DOI: 10.1039/c6nr00711b

Growth factor-functionalized silk membranes support wound healing in vitro.

PubMed

Bienert, M; Hoss, M; Bartneck, M; Weinandy, S; Böbel, M; Jockenhövel, S; Knüchel, R; Pottbacker, K; Wöltje, M; Jahnen-Dechent, W; Neuss, S

2017-08-16

Chronic wounds represent a serious problem in daily medical routine requiring improved wound care. Silk of the domesticated silkworm (Bombyx mori) has been used to form a variety of biomaterials for medical applications. We genetically engineered B. mori to produce silk functionalized with growth factors to promote wound healing in vitro. In this study FGF-, EGF-, KGF-, PDGF- or VEGF-functionalized silk membranes were compared to native B. mori silk membranes without growth factors for their ability to support wound healing in vitro. All silk membranes were cytocompatible and supported macrophage secretion of neutrophil recruiting factor CXCL1 and monocyte chemoattractant protein 1 (MCP-1). VEGF-functionalized silk significantly outperformed other growth factor-functionalized silk membranes, but not native silk in angiogenesis assays. In addition, EGF- and VEGF-functionalized silk membranes slightly enhanced macrophage adhesion compared to silk without growth factors. In wound healing assays in vitro (reduction of wound lesion), dermal equivalents showed a higher wound healing capacity when covered with EGF-, FGF- or VEGF-functionalized silk membranes compared to native, KGF- or PDGF-functionalized silk membranes. Keratinocyte migration and growth is overstimulated by KGF- and VEGF-functionalized silk membranes. In conclusion, growth factor-functionalized silk membranes prepared from genetically engineered silk worm glands are promising wound dressings for future wound healing therapies.

Analyzing Protein Clusters on the Plasma Membrane: Application of Spatial Statistical Analysis Methods on Super-Resolution Microscopy Images.

PubMed

Paparelli, Laura; Corthout, Nikky; Pavie, Benjamin; Annaert, Wim; Munck, Sebastian

2016-01-01

The spatial distribution of proteins within the cell affects their capability to interact with other molecules and directly influences cellular processes and signaling. At the plasma membrane, multiple factors drive protein compartmentalization into specialized functional domains, leading to the formation of clusters in which intermolecule interactions are facilitated. Therefore, quantifying protein distributions is a necessity for understanding their regulation and function. The recent advent of super-resolution microscopy has opened up the possibility of imaging protein distributions at the nanometer scale. In parallel, new spatial analysis methods have been developed to quantify distribution patterns in super-resolution images. In this chapter, we provide an overview of super-resolution microscopy and summarize the factors influencing protein arrangements on the plasma membrane. Finally, we highlight methods for analyzing clusterization of plasma membrane proteins, including examples of their applications.

Organelle Communication at Membrane Contact Sites (MCS): From Curiosity to Center Stage in Cell Biology and Biomedical Research.

PubMed

Simmen, Thomas; Tagaya, Mitsuo

2017-01-01

Cell biology has long recognized that organelles can communicate with each other. Initially, such communication was thought to occur primarily via vesicular trafficking between biochemically distinct organelles. However, studies starting in the 1970s on lipid metabolism have unearthed another way how organelles can communicate and have spawned the field of membrane contact sites (MCS). While, initially, MCS had been recognized as fluid entities that mediate lipid and ion transport in an ad hoc manner, more recently MCS have been found to depend on protein-protein interactions that control themselves a variety of MCS functions. As a result, the cell biological definition of an intracellular organelle as an isolated membrane compartment is now being revised. Accordingly, the organelle definition now describes organelles as dynamic membrane compartments that function in a milieu of coordinated contacts with other organelles. Through these mercurial functions, MCS dictate the function of organelles to a large extent but also play important roles in a number of diseases, including type 2 diabetes, neurodegenerative diseases, infections, and cancer. This book assembles reviews that describe our quickly evolving knowledge about organellar communication on MCS and the significance of MCS for disease.

Plant Endoplasmic Reticulum-Plasma Membrane Contact Sites.

PubMed

Wang, Pengwei; Hawes, Chris; Hussey, Patrick J

2017-04-01

The endoplasmic reticulum (ER) acts as a superhighway with multiple sideroads that connects the different membrane compartments including the ER to the plasma membrane (PM). ER-PM contact sites (EPCSs) are a common feature in eukaryotic organisms, but have not been studied well in plants owing to the lack of molecular markers and to the difficulty in resolving the EPCS structure using conventional microscopy. Recently, however, plant protein complexes required for linking the ER and PM have been identified. This is a further step towards understanding the structure and function of plant EPCSs. We highlight some recent studies in this field and suggest several hypotheses that relate to the possible function of EPCSs in plants. Copyright © 2016. Published by Elsevier Ltd.

3D-printed membrane for guided tissue regeneration.

PubMed

Tayebi, Lobat; Rasoulianboroujeni, Morteza; Moharamzadeh, Keyvan; Almela, Thafar K D; Cui, Zhanfeng; Ye, Hua

2018-03-01

Three-dimensional (3D) printing is currently being intensely studied for a diverse set of applications, including the development of bioengineered tissues, as well as the production of functional biomedical materials and devices for dental and orthopedic applications. The aim of this study was to develop and characterize a 3D-printed hybrid construct that can be potentially suitable for guided tissue regeneration (GTR). For this purpose, the rheology analyses have been performed on different bioinks and a specific solution comprising 8% gelatin, 2% elastin and 0.5% sodium hyaluronate has been selected as the most suitable composition for printing a structured membrane for GTR application. Each membrane is composed of 6 layers with strand angles from the first layer to the last layer of 45, 135, 0, 90, 0 and 90°. Confirmed by 3D Laser Measuring imaging, the membrane has small pores on one side and large pores on the other to be able to accommodate different cells like osteoblasts, fibroblasts and keratinocytes on different sides. The ultimate cross-linked product is a 150μm thick flexible and bendable membrane with easy surgical handling. Static and dynamic mechanical testing revealed static tensile modules of 1.95±0.55MPa and a dynamic tensile storage modulus of 314±50kPa. Through seeding the membranes with fibroblast and keratinocyte cells, the results of in vitro tests, including histological analysis, tissue viability examinations and DAPI staining, indicated that the membrane has desirable in vitro biocompatibility. The membrane has demonstrated the barrier function of a GTR membrane by thorough separation of the oral epithelial layer from the underlying tissues. In conclusion, we have characterized a biocompatible and bio-resorbable 3D-printed structured gelatin/elastin/sodium hyaluronate membrane with optimal biostability, mechanical strength and surgical handling characteristics in terms of suturability for potential application in GTR procedures. Copyright © 2017 Elsevier B.V. All rights reserved.

A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro.

PubMed

Shadforth, Audra M A; Suzuki, Shuko; Theodoropoulos, Christina; Richardson, Neil A; Chirila, Traian V; Harkin, Damien G

2017-06-01

Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 µm). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4 months. Control cultures were grown on polyester cell culture well inserts (Transwell ® ). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12 weeks. Immunocytochemistry at 16 weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na + /K + -ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Biochemistry and Biophysics of HIV-1 gp41 – membrane interactions

PubMed Central

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-01-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein – mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), N-terminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors. PMID:22044229

Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

PubMed

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-12-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

Rewiring of cellular membrane homeostasis by picornaviruses.

PubMed

Belov, George A; Sztul, Elizabeth

2014-09-01

Viruses are obligatory intracellular parasites and utilize host elements to support key viral processes, including penetration of the plasma membrane, initiation of infection, replication, and suppression of the host's antiviral defenses. In this review, we focus on picornaviruses, a family of positive-strand RNA viruses, and discuss the mechanisms by which these viruses hijack the cellular machinery to form and operate membranous replication complexes. Studies aimed at revealing factors required for the establishment of viral replication structures identified several cellular-membrane-remodeling proteins and led to the development of models in which the virus used a preexisting cellular-membrane-shaping pathway "as is" for generating its replication organelles. However, as more data accumulate, this view is being increasingly questioned, and it is becoming clearer that viruses may utilize cellular factors in ways that are distinct from the normal functions of these proteins in uninfected cells. In addition, the proteincentric view is being supplemented by important new studies showing a previously unappreciated deep remodeling of lipid homeostasis, including extreme changes to phospholipid biosynthesis and cholesterol trafficking. The data on viral modifications of lipid biosynthetic pathways are still rudimentary, but it appears once again that the viruses may rewire existing pathways to generate novel functions. Despite remarkable progress, our understanding of how a handful of viral proteins can completely overrun the multilayered, complex mechanisms that control the membrane organization of a eukaryotic cell remains very limited. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Whole-cell patch clamp recording of voltage-sensitive Ca²+ channel currents: heterologous expression systems and dissociated brain neurons.

PubMed

Hainsworth, Atticus H; Randall, Andrew D; Stefani, Alessandro

2005-01-01

Voltage-sensitive Ca(2+) channels (VSCC) play a central role in an extensive array of physiological processes. Their importance in cellular function arises from their ability both to sense membrane voltage and to conduct Ca(2+) ions, two facets that couple membrane excitability to a key intracellular second messenger. Through this relationship, activation of VSCCs is tightly coupled to the gamut of cellular functions dependent on intracellular Ca(2+), including muscle contraction, energy metabolism, gene expression, and exocytotic/endocytotic cycling.

A cell-free translocation system using extracts of cultured insect cells to yield functional membrane proteins.

PubMed

Ezure, Toru; Nanatani, Kei; Sato, Yoko; Suzuki, Satomi; Aizawa, Keishi; Souma, Satoshi; Ito, Masaaki; Hohsaka, Takahiro; von Heijine, Gunnar; Utsumi, Toshihiko; Abe, Keietsu; Ando, Eiji; Uozumi, Nobuyuki

2014-01-01

Cell-free protein synthesis is a powerful method to explore the structure and function of membrane proteins and to analyze the targeting and translocation of proteins across the ER membrane. Developing a cell-free system based on cultured cells for the synthesis of membrane proteins could provide a highly reproducible alternative to the use of tissues from living animals. We isolated Sf21 microsomes from cultured insect cells by a simplified isolation procedure and evaluated the performance of the translocation system in combination with a cell-free translation system originating from the same source. The isolated microsomes contained the basic translocation machinery for polytopic membrane proteins including SRP-dependent targeting components, translocation channel (translocon)-dependent translocation, and the apparatus for signal peptide cleavage and N-linked glycosylation. A transporter protein synthesized with the cell-free system could be functionally reconstituted into a lipid bilayer. In addition, single and double labeling with non-natural amino acids could be achieved at both the lumen side and the cytosolic side in this system. Moreover, tail-anchored proteins, which are post-translationally integrated by the guided entry of tail-anchored proteins (GET) machinery, were inserted correctly into the microsomes. These results showed that the newly developed cell-free translocation system derived from cultured insect cells is a practical tool for the biogenesis of properly folded polytopic membrane proteins as well as tail-anchored proteins.

SChloro: directing Viridiplantae proteins to six chloroplastic sub-compartments.

PubMed

Savojardo, Castrense; Martelli, Pier Luigi; Fariselli, Piero; Casadio, Rita

2017-02-01

Chloroplasts are organelles found in plants and involved in several important cell processes. Similarly to other compartments in the cell, chloroplasts have an internal structure comprising several sub-compartments, where different proteins are targeted to perform their functions. Given the relation between protein function and localization, the availability of effective computational tools to predict protein sub-organelle localizations is crucial for large-scale functional studies. In this paper we present SChloro, a novel machine-learning approach to predict protein sub-chloroplastic localization, based on targeting signal detection and membrane protein information. The proposed approach performs multi-label predictions discriminating six chloroplastic sub-compartments that include inner membrane, outer membrane, stroma, thylakoid lumen, plastoglobule and thylakoid membrane. In comparative benchmarks, the proposed method outperforms current state-of-the-art methods in both single- and multi-compartment predictions, with an overall multi-label accuracy of 74%. The results demonstrate the relevance of the approach that is eligible as a good candidate for integration into more general large-scale annotation pipelines of protein subcellular localization. The method is available as web server at http://schloro.biocomp.unibo.it gigi@biocomp.unibo.it.

Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

NASA Astrophysics Data System (ADS)

Kamiya, Koki; Kawano, Ryuji; Osaki, Toshihisa; Akiyoshi, Kazunari; Takeuchi, Shoji

2016-09-01

Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

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.

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.

Theory of Ion and Water Transport in Reverse-Osmosis Membranes

NASA Astrophysics Data System (ADS)

Oren, Y. S.; Biesheuvel, P. M.

2018-02-01

We present a theory for ion and water transport through reverse-osmosis (RO) membranes based on a Maxwell-Stefan framework combined with hydrodynamic theory for the reduced motion of particles in thin pores. We take into account all driving forces and frictions both on the fluid (water) and on the ions including ion-fluid friction and ion-wall friction. By including the acid-base characteristic of the carbonic acid system, the boric acid system, H3O+/OH- , and the membrane charge, we locally determine p H , the effective charge of the membrane, and the dissociation degree of carbonic acid and boric acid. We present calculation results for an experiment with fixed feed concentration, where effluent composition is a self-consistent function of fluxes through the membrane. A comparison with experimental results from literature for fluid flow vs pressure, and for salt and boron rejection, shows that our theory agrees very well with the available data. Our model is based on realistic assumptions for the effective size of the ions and makes use of a typical pore size of a commercial RO membrane.

Structural and functional changes in acute liver injury.

PubMed Central

Smuckler, E A

1976-01-01

Carbon tetrachloride produces liver cell injury in a variety of animal species. The first structurally recognizable changes occur in the endoplasmic reticulum, with alteration in ribosome-membrane interactions. Later there is an increase in intracellular fat, and the formation of tangled nets of the ergastoplasm. At no time are there changes in mitochondria or single membrane limited bodies in cells with intact plasmalemma, although a relative increase in cell sap may appear. In dead cells (those with plasmalemma discontinuties) crystalline deposits of calcium phosphatase may be noted. Functional changes are related to the endoplasmic reticulum and the plasma membrane. An early decrease in protein synthesis takes place; an accumulation of neutral lipid is related to this change. Later alterations in the ergastoplasmic functions (e.g., mixed function oxidation) occurs. Carbon tetrachloride is not the active agent; rather, a product of its metabolism, probably the CC1, free radical, is. The mechanisms of injury include macromolecular adduction and peroxide propagation. A third possibility includes a cascade effect with the production of secondary and tertiary products, also toxic in nature, with the ability to produce more widespread damage to intracellular structures. Images FIGURE 1. FIGURE 2. FIGURE 3. FIGURE 4. FIGURE 5. FIGURE 6. FIGURE 7. FIGURE 11. PMID:1001290

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.

Unconventional Transport Routes of Soluble and Membrane Proteins and Their Role in Developmental Biology

PubMed Central

Pompa, Andrea; De Marchis, Francesca; Pallotta, Maria Teresa; Benitez-Alfonso, Yoselin; Jones, Alexandra; Schipper, Kerstin; Moreau, Kevin; Žárský, Viktor; Di Sansebastiano, Gian Pietro; Bellucci, Michele

2017-01-01

Many proteins and cargoes in eukaryotic cells are secreted through the conventional secretory pathway that brings proteins and membranes from the endoplasmic reticulum to the plasma membrane, passing through various cell compartments, and then the extracellular space. The recent identification of an increasing number of leaderless secreted proteins bypassing the Golgi apparatus unveiled the existence of alternative protein secretion pathways. Moreover, other unconventional routes for secretion of soluble or transmembrane proteins with initial endoplasmic reticulum localization were identified. Furthermore, other proteins normally functioning in conventional membrane traffic or in the biogenesis of unique plant/fungi organelles or in plasmodesmata transport seem to be involved in unconventional secretory pathways. These alternative pathways are functionally related to biotic stress and development, and are becoming more and more important in cell biology studies in yeast, mammalian cells and in plants. The city of Lecce hosted specialists working on mammals, plants and microorganisms for the inaugural meeting on “Unconventional Protein and Membrane Traffic” (UPMT) during 4–7 October 2016. The main aim of the meeting was to include the highest number of topics, summarized in this report, related to the unconventional transport routes of protein and membranes. PMID:28346345

Caveolins and caveolae in ocular physiology and pathophysiology.

PubMed

Gu, Xiaowu; Reagan, Alaina M; McClellan, Mark E; Elliott, Michael H

2017-01-01

Caveolae are specialized, invaginated plasma membrane domains that are defined morphologically and by the expression of signature proteins called, caveolins. Caveolae and caveolins are abundant in a variety of cell types including vascular endothelium, glia, and fibroblasts where they play critical roles in transcellular transport, endocytosis, mechanotransduction, cell proliferation, membrane lipid homeostasis, and signal transduction. Given these critical cellular functions, it is surprising that ablation of the caveolae organelle does not result in lethality suggesting instead that caveolae and caveolins play modulatory roles in cellular homeostasis. Caveolar components are also expressed in ocular cell types including retinal vascular cells, Müller glia, retinal pigment epithelium (RPE), conventional aqueous humor outflow cells, the corneal epithelium and endothelium, and the lens epithelium. In the eye, studies of caveolae and other membrane microdomains (i.e., "lipid rafts") have lagged behind what is a substantial body of literature outside vision science. However, interest in caveolae and their molecular components has increased with accumulating evidence of important roles in vision-related functions such as blood-retinal barrier homeostasis, ocular inflammatory signaling, pathogen entry at the ocular surface, and aqueous humor drainage. The recent association of CAV1/2 gene loci with primary open angle glaucoma and intraocular pressure has further enhanced the need to better understand caveolar functions in the context of ocular physiology and disease. Herein, we provide the first comprehensive review of literature on caveolae, caveolins, and other membrane domains in the context of visual system function. This review highlights the importance of caveolae domains and their components in ocular physiology and pathophysiology and emphasizes the need to better understand these important modulators of cellular function. Copyright © 2016 Elsevier Ltd. All rights reserved.

Caveolins and caveolae in ocular physiology and pathophysiology

PubMed Central

Gu, Xiaowu; Reagan, Alaina M.; McClellan, Mark E.; Elliott, Michael H.

2016-01-01

Caveolae are specialized, invaginated plasma membrane domains that are defined morphologically and by the expression of signature proteins called, caveolins. Caveolae and caveolins are abundant in a variety of cell types including vascular endothelium, glia, and fibroblasts where they play critical roles in transcellular transport, endocytosis, mechanotransduction, cell proliferation, membrane lipid homeostasis, and signal transduction. Given these critical cellular functions, it is surprising that ablation of the caveolae organelle does not result in lethality suggesting instead that caveolae and caveolins play modulatory roles in cellular homeostasis. Caveolar components are also expressed in ocular cell types including retinal vascular cells, Müller glia, retinal pigment epithelium (RPE), conventional aqueous humor outflow cells, the corneal epithelium and endothelium, and the lens epithelium. In the eye, studies of caveolae and other membrane microdomains (i.e., “lipid rafts”) have lagged behind what is a substantial body of literature outside vision science. However, interest in caveolae and their molecular components has increased with accumulating evidence of important roles in vision-related functions such as blood-retinal barrier homeostasis, ocular inflammatory signalling, pathogen entry at the ocular surface, and aqueous humor drainage. The recent association of CAV1/2 gene loci with primary open angle glaucoma and intraocular pressure has further enhanced the need to better understand caveolar functions in the context of ocular physiology and disease. Herein, we provide the first comprehensive review of literature on caveolae, caveolins, and other membrane domains in the context of visual system function. This review highlights the importance of caveolae domains and their components in ocular physiology and pathophysiology and emphasizes the need to better understand these important modulators of cellular function. PMID:27664379

Membrane Composition Tunes the Outer Hair Cell Motor

NASA Astrophysics Data System (ADS)

Rajagopalan, L.; Sfondouris, J.; Oghalai, J. S.; Pereira, F. A.; Brownell, W. E.

2009-02-01

Cholesterol and docosahexaenoic acid (DHA), an ω-3 fatty acid, affect membrane mechanical properties in different ways and modulate the function of membrane proteins. We have probed the functional consequence of altering cholesterol and DHA levels in the membranes of OHCs and prestin expressing HEK cells. Large, dynamic and reversible changes in prestin-associated charge movement and OHC motor activity result from altering the concentration of membrane cholesterol. Increasing membrane cholesterol shifts the q/V function ~ 50 mV in the hyperpolarizing direction, possibly a response related to increases in membrane stiffness. The voltage shift is linearly related to total membrane cholesterol. Increasing cholesterol also decreases the total charge moved in a linear fashion. Decreasing membrane cholesterol shifts the q/V function ~ 50 mV in the depolarizing direction with little or no effect on the amount of charge moved. In vivo increases in membrane cholesterol transiently increase but ultimately lead to decreases in DPOAE. Docosahexaenoic acid shifts the q/V function in the hyperpolarizing direction < 15 mV and increases total charge moved. Tuning of cochlear function by membrane cholesterol contributes to the exquisite temporal and frequency processing of mammalian hearing by optimizing the cochlear amplifier.

The membrane proteome of Medicago truncatula roots displays qualitative and quantitative changes in response to arbuscular mycorrhizal symbiosis.

PubMed

Abdallah, Cosette; Valot, Benoit; Guillier, Christelle; Mounier, Arnaud; Balliau, Thierry; Zivy, Michel; van Tuinen, Diederik; Renaut, Jenny; Wipf, Daniel; Dumas-Gaudot, Eliane; Recorbet, Ghislaine

2014-08-28

Arbuscular mycorrhizal (AM) symbiosis that associates roots of most land plants with soil-borne fungi (Glomeromycota), is characterized by reciprocal nutritional benefits. Fungal colonization of plant roots induces massive changes in cortical cells where the fungus differentiates an arbuscule, which drives proliferation of the plasma membrane. Despite the recognized importance of membrane proteins in sustaining AM symbiosis, the root microsomal proteome elicited upon mycorrhiza still remains to be explored. In this study, we first examined the qualitative composition of the root membrane proteome of Medicago truncatula after microsome enrichment and subsequent in depth analysis by GeLC-MS/MS. The results obtained highlighted the identification of 1226 root membrane protein candidates whose cellular and functional classifications predispose plastids and protein synthesis as prevalent organelle and function, respectively. Changes at the protein abundance level between the membrane proteomes of mycorrhizal and nonmycorrhizal roots were further monitored by spectral counting, which retrieved a total of 96 proteins that displayed a differential accumulation upon AM symbiosis. Besides the canonical markers of the periarbuscular membrane, new candidates supporting the importance of membrane trafficking events during mycorrhiza establishment/functioning were identified, including flotillin-like proteins. The data have been deposited to the ProteomeXchange with identifier PXD000875. During arbuscular mycorrhizal symbiosis, one of the most widespread mutualistic associations in nature, the endomembrane system of plant roots is believed to undergo qualitative and quantitative changes in order to sustain both the accommodation process of the AM fungus within cortical cells and the exchange of nutrients between symbionts. Large-scale GeLC-MS/MS proteomic analysis of the membrane fractions from mycorrhizal and nonmycorrhizal roots of M. truncatula coupled to spectral counting retrieved around one hundred proteins that displayed changes in abundance upon mycorrhizal establishment. The symbiosis-related membrane proteins that were identified mostly function in signaling/membrane trafficking and nutrient uptake regulation. Besides extending the coverage of the root membrane proteome of M. truncatula, new candidates involved in the symbiotic program emerged from the current study, which pointed out a dynamic reorganization of microsomal proteins during the accommodation of AM fungi within cortical cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Graphene-based membranes.

PubMed

Liu, Gongping; Jin, Wanqin; Xu, Nanping

2015-08-07

Graphene is a well-known two-dimensional material that exhibits preeminent electrical, mechanical and thermal properties owing to its unique one-atom-thick structure. Graphene and its derivatives (e.g., graphene oxide) have become emerging nano-building blocks for separation membranes featuring distinct laminar structures and tunable physicochemical properties. Extraordinary molecular separation properties for purifying water and gases have been demonstrated by graphene-based membranes, which have attracted a huge surge of interest during the past few years. This tutorial review aims to present the latest groundbreaking advances in both the theoretical and experimental chemical science and engineering of graphene-based membranes, including their design, fabrication and application. Special attention will be given to the progresses in processing graphene and its derivatives into separation membranes with three distinct forms: a porous graphene layer, assembled graphene laminates and graphene-based composites. Moreover, critical views on separation mechanisms within graphene-based membranes will be provided based on discussing the effect of inter-layer nanochannels, defects/pores and functional groups on molecular transport. Furthermore, the separation performance of graphene-based membranes applied in pressure filtration, pervaporation and gas separation will be summarized. This article is expected to provide a compact source of relevant and timely information and will be of great interest to all chemists, physicists, materials scientists, engineers and students entering or already working in the field of graphene-based membranes and functional films.

Lateral Membrane Waves Constitute a Universal Dynamic Pattern of Motile Cells

NASA Astrophysics Data System (ADS)

Döbereiner, Hans-Günther; Dubin-Thaler, Benjamin J.; Hofman, Jake M.; Xenias, Harry S.; Sims, Tasha N.; Giannone, Grégory; Dustin, Michael L.; Wiggins, Chris H.; Sheetz, Michael P.

2006-07-01

We have monitored active movements of the cell circumference on specifically coated substrates for a variety of cells including mouse embryonic fibroblasts and T cells, as well as wing disk cells from fruit flies. Despite having different functions and being from multiple phyla, these cell types share a common spatiotemporal pattern in their normal membrane velocity; we show that protrusion and retraction events are organized in lateral waves along the cell membrane. These wave patterns indicate both spatial and temporal long-range periodic correlations of the actomyosin gel.

The Function of V-ATPases in Cancer

PubMed Central

Stransky, Laura; Cotter, Kristina

2016-01-01

The vacuolar ATPases (V-ATPases) are a family of proton pumps that couple ATP hydrolysis to proton transport into intracellular compartments and across the plasma membrane. They function in a wide array of normal cellular processes, including membrane traffic, protein processing and degradation, and the coupled transport of small molecules, as well as such physiological processes as urinary acidification and bone resorption. The V-ATPases have also been implicated in a number of disease processes, including viral infection, renal disease, and bone resorption defects. This review is focused on the growing evidence for the important role of V-ATPases in cancer. This includes functions in cellular signaling (particularly Wnt, Notch, and mTOR signaling), cancer cell survival in the highly acidic environment of tumors, aiding the development of drug resistance, as well as crucial roles in tumor cell invasion, migration, and metastasis. Of greatest excitement is evidence that at least some tumors express isoforms of V-ATPase subunits whose disruption is not lethal, leading to the possibility of developing anti-cancer therapeutics that selectively target V-ATPases that function in cancer cells. PMID:27335445

New Aspects of the Contribution of ER to SOCE Regulation: TRPC Proteins as a Link Between Plasma Membrane Ion Transport and Intracellular Ca2+ Stores.

PubMed

Bavencoffe, Alexis; Zhu, Michael Xi; Tian, Jin-Bin

2017-01-01

Transient receptor potential canonical (TRPC) proteins were identified as molecular candidates of receptor- and/or store-operated channels because of their close homology to the Drosophila TRP and TRPL. Functional studies have revealed that TRPC channels play an integrated part of phospholipase C-transduced cell signaling, mediating the influx of both Ca 2+ and Na + into cells. As a consequence, the TRPC channels have diverse functional roles in different cell types, including metabotropic receptor-evoked membrane depolarization and intracellular Ca 2+ concentration elevation. Depending on the cellular environment and the protein partners present in the channel complex, the TRPC channels display different biophysical properties and mechanisms of regulation, including but not limited to the Ca 2+ filling state of the endoplasmic reticulum. Despite the overwhelming focus on STIM-regulated Orai channels for store-operated Ca 2+ entry, evidence is growing for STIM-operated TRPC channel activities in various cell types, demonstrating both store-dependent and store-independent mechanisms of TRPC channel gating. The existence of physical and functional interactions between plasma membrane-localized TRPC channels and other proteins involved in sensing and regulating the intracellular Ca 2+ store contents, such as inositol trisphosphate receptors, Junctate, and Homer, further argues for the role of TRPC proteins in linking plasma membrane ion transport with intracellular Ca 2+ stores. The interplay among these proteins will likely define the functional significance of TRPC channel activation in different cellular contexts and under different modes of stimulations.

The three lives of viral fusion peptides

PubMed Central

Apellániz, Beatriz; Huarte, Nerea; Largo, Eneko; Nieva, José L.

2014-01-01

Fusion peptides comprise conserved hydrophobic domains absolutely required for the fusogenic activity of glycoproteins from divergent virus families. After 30 years of intensive research efforts, the structures and functions underlying their high degree of sequence conservation are not fully elucidated. The long-hydrophobic viral fusion peptide (VFP) sequences are structurally constrained to access three successive states after biogenesis. Firstly, the VFP sequence must fulfill the set of native interactions required for (meta) stable folding within the globular ectodomains of glycoprotein complexes. Secondly, at the onset of the fusion process, they get transferred into the target cell membrane and adopt specific conformations therein. According to commonly accepted mechanistic models, membrane-bound states of the VFP might promote the lipid bilayer remodeling required for virus-cell membrane merger. Finally, at least in some instances, several VFPs co-assemble with transmembrane anchors into membrane integral helical bundles, following a locking movement hypothetically coupled to fusion-pore expansion. Here we review different aspects of the three major states of the VFPs, including the functional assistance by other membrane-transferring glycoprotein regions, and discuss briefly their potential as targets for clinical intervention. PMID:24704587

Transport dynamics in membranes of photosynthetic purple bacteria

NASA Astrophysics Data System (ADS)

Caycedo, Felipe; Rodriguez, Ferney; Quiroga, Luis; Fassioli, Francesca; Johnson, Neil

2007-03-01

Photo-Syntethic Unit (PSU) of purple bacteria is conformed by three basic constituents: Light Harvesting Complex 2 (LH2) antenna complexes, where chromophores are distributed in a ring in close contact with caroteniods with a function of collecting light; LH1s, ring shaped structures of chromophores which harvest and funnel excitations to the Reaction Centre (RC), where phtosynthesis takes place. Studies concerning a single PSU have been capable of reproducing experimental transfer times, but incapable of explaining the fact that architecture LH2-LH1-RC of phototosynthetic membranes changes as light intensity conditions vary. The organization of antenna complexes in the membranes that support PSU seems to have its own functionality. A hopping model where excitations are transferred within a membrane is used, and populations of RC, LH1 and LH2 are investigated. Different statistics concerning arrival times of excitations that excite a single PSU are considered and compared with the global model where coordinates of a great portion of a membrane are included. The model permits in a classical basis to understand which parameters make photosynthesis in purple bateria efficient and reliable.

Stresses in curved nematic membranes.

PubMed

Santiago, J A

2018-05-01

Ordering configurations of a director field on a curved membrane induces stress. In this work, we present a theoretical framework to calculate the stress tensor and the torque as a consequence of the nematic ordering; we use the variational principle and invariance of the energy under Euclidean motions. Euler-Lagrange equations of the membrane as well as the corresponding boundary conditions also appear as natural results. The stress tensor found includes attraction-repulsion forces between defects; likewise, defects are attracted to patches with the same sign in Gaussian curvature. These forces are mediated by the Green function of the Laplace-Beltrami operator of the surface. In addition, we find nonisotropic forces that involve derivatives of the Green function and the Gaussian curvature, even in the normal direction to the membrane. We examine the case of axial membranes to analyze the spherical one. For spherical vesicles we find the modified Young-Laplace law as a consequence of the nematic texture. In the case of spherical cap with defect at the north pole, we find that the force is repulsive with respect to the north pole, indicating that it is an unstable equilibrium point.

The sigma-1 receptor chaperone as an inter-organelle signaling modulator

PubMed Central

Su, Tsung-Ping; Hayashi, Teruo; Maurice, Tangui; Buch, Shilpa; Ruoho, Arnold E.

2010-01-01

Inter-organelle signaling plays important roles in many physiological functions. Endoplasmic reticulum (ER)-mitochondrion signaling affects intra-mitochondrial calcium (Ca2+) homeostasis and cellular bioenergetics. ER-nucleus signaling attenuates ER stress. ER-plasma membrane signaling regulates cytosolic Ca2+ homeostasis, and ER-mitochondrion-plasma membrane signaling regulates hippocampal dendritic spine formation. Here we propose that the sigma-1 receptor (Sig-1R), an ER chaperone protein, acts as an inter-organelle signaling modulator. Sig-1Rs normally reside at the ER-mitochondrion contact called the MAM (mitochondrion-associated ER membrane), where Sig-1Rs regulate ER-mitochondrion signaling and the ER-nucleus cross-talk. When cells are stimulated by ligands or undergo prolonged stress, Sig-1Rs translocate from the MAM to the ER reticular network and plasmalemma/plasma membrane to regulate a variety of functional proteins, including ion channels, receptors, and kinases. Thus, the Sig-1R serves as an inter-organelle signaling modulator locally at the MAM and remotely at the plasmalemma/plasma membrane. Many pharmacological/physiological effects of Sig-1Rs may relate to this unique action of Sig-1Rs. PMID:20869780

Stresses in curved nematic membranes

NASA Astrophysics Data System (ADS)

Santiago, J. A.

2018-05-01

Ordering configurations of a director field on a curved membrane induces stress. In this work, we present a theoretical framework to calculate the stress tensor and the torque as a consequence of the nematic ordering; we use the variational principle and invariance of the energy under Euclidean motions. Euler-Lagrange equations of the membrane as well as the corresponding boundary conditions also appear as natural results. The stress tensor found includes attraction-repulsion forces between defects; likewise, defects are attracted to patches with the same sign in Gaussian curvature. These forces are mediated by the Green function of the Laplace-Beltrami operator of the surface. In addition, we find nonisotropic forces that involve derivatives of the Green function and the Gaussian curvature, even in the normal direction to the membrane. We examine the case of axial membranes to analyze the spherical one. For spherical vesicles we find the modified Young-Laplace law as a consequence of the nematic texture. In the case of spherical cap with defect at the north pole, we find that the force is repulsive with respect to the north pole, indicating that it is an unstable equilibrium point.

RELATIVE POTENCIES FOR ACUTE EFFECTS OF PYRETHROIDS ON MOTOR FUNCTION IN RATS.

EPA Science Inventory

A proposed common mode-of-action for pyrethroid insecticides, includes alterations in sodium channel dynamics in nervous system tissues, consequent disturbance of neuronal membrane polarization, abnormal discharge in targeted neurons, and changes in nervous system function. The p...

Profiling of the Tox21 Chemical Collection for Mitochondrial Function: I. Compounds that Decrease Mitochondrial Membrane Potential

EPA Science Inventory

Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding how different environmental chemicals and drug-like molecules impact mitochondrial function rep...

Photoreactive synthetic regulator of protein function and methods of use thereof

DOEpatents

Trauner, Dirk; Isacoff, Ehud Y; Kramer, Richard H; Banghart, Matthew R; Fortin, Doris L; Mourot, Alexandre

2015-03-31

The present disclosure provides a photoreactive synthetic regulator of protein function. The present disclosure further provides a light-regulated polypeptide that includes a subject synthetic regulator. Also provided are cells and membranes comprising a subject light-regulated polypeptide. The present disclosure further provides methods of modulating protein function, involving use of light.

Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

PubMed Central

Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

1998-01-01

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

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.

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.

Preparation of membrane rafts.

PubMed

Waugh, Mark G; Hsuan, J Justin

2009-01-01

The concept that biological membranes contain microdomains of specialized lipid and protein composition has attracted great attention in recent years. Initially, the focus in the field was very much on the characterization of cholesterol-and sphingolipid-rich plasma membrane microdomains that were resistant to solubilization in the cold non-ionic detergent Triton X-100. Such detergent-insoluble membrane domains were of low buoyant density and could be readily purified on sucrose equilibrium density gradients. The intrinsic buoyancy of the detergent-insoluble domains gave rise to the term "lipid rafts." Cholesterol- and sphingolipid-rich rafts at the plasma membrane have been implicated in a wide range of cellular processes, including pathogen invasion, receptor signaling, and endocytosis. However, work with other non-ionic detergents such as Lubrol WX and Brij-98 has revealed the existence of various raft subtypes with differing lipid compositions and proposed functions. More recently, there has been some focus on isolating lipid rafts from intracellular organelles, in particular membranes from the Golgi-endosomal pathway, where raft lipids have been proposed to function in processes such as the sorting of vesicular cargo and the processing of amyloid precursor protein. While there remains a large degree of controversy surrounding the purity, the physiological importance, and even the existence of different types of lipid rafts in intact cells, the ability to routinely purify such domains has led to significant progress in understanding the functional architecture of biological membranes. We describe a number of widely used methods to prepare rafts, based on early preparations of caveolae by density gradient ultracentrifugation and immunoaffinity precipitation.

Mapping membrane activity in undiscovered peptide sequence space using machine learning

PubMed Central

Fulan, Benjamin M.; Wong, Gerard C. L.

2016-01-01

There are some ∼1,100 known antimicrobial peptides (AMPs), which permeabilize microbial membranes but have diverse sequences. Here, we develop a support vector machine (SVM)-based classifier to investigate ⍺-helical AMPs and the interrelated nature of their functional commonality and sequence homology. SVM is used to search the undiscovered peptide sequence space and identify Pareto-optimal candidates that simultaneously maximize the distance σ from the SVM hyperplane (thus maximize its “antimicrobialness”) and its ⍺-helicity, but minimize mutational distance to known AMPs. By calibrating SVM machine learning results with killing assays and small-angle X-ray scattering (SAXS), we find that the SVM metric σ correlates not with a peptide’s minimum inhibitory concentration (MIC), but rather its ability to generate negative Gaussian membrane curvature. This surprising result provides a topological basis for membrane activity common to AMPs. Moreover, we highlight an important distinction between the maximal recognizability of a sequence to a trained AMP classifier (its ability to generate membrane curvature) and its maximal antimicrobial efficacy. As mutational distances are increased from known AMPs, we find AMP-like sequences that are increasingly difficult for nature to discover via simple mutation. Using the sequence map as a discovery tool, we find a unexpectedly diverse taxonomy of sequences that are just as membrane-active as known AMPs, but with a broad range of primary functions distinct from AMP functions, including endogenous neuropeptides, viral fusion proteins, topogenic peptides, and amyloids. The SVM classifier is useful as a general detector of membrane activity in peptide sequences. PMID:27849600

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.

Determining the Orientation and Localization of Membrane-Bound Peptides

PubMed Central

Hohlweg, Walter; Kosol, Simone; Zangger, Klaus

2012-01-01

Many naturally occurring bioactive peptides bind to biological membranes. Studying and elucidating the mode of interaction is often an essential step to understand their molecular and biological functions. To obtain the complete orientation and immersion depth of such compounds in the membrane or a membrane-mimetic system, a number of methods are available, which are separated in this review into four main classes: solution NMR, solid-state NMR, EPR and other methods. Solution NMR methods include the Nuclear Overhauser Effect (NOE) between peptide and membrane signals, residual dipolar couplings and the use of paramagnetic probes, either within the membrane-mimetic or in the solvent. The vast array of solid state NMR methods to study membrane-bound peptide orientation and localization includes the anisotropic chemical shift, PISA wheels, dipolar waves, the GALA, MAOS and REDOR methods and again the use of paramagnetic additives on relaxation rates. Paramagnetic additives, with their effect on spectral linewidths, have also been used in EPR spectroscopy. Additionally, the orientation of a peptide within a membrane can be obtained by the anisotropic hyperfine tensor of a rigidly attached nitroxide label. Besides these magnetic resonance techniques a series of other methods to probe the orientation of peptides in membranes has been developed, consisting of fluorescence-, infrared- and oriented circular dichroism spectroscopy, colorimetry, interface-sensitive X-ray and neutron scattering and Quartz crystal microbalance. PMID:22044140

Lipid functions in cytochrome bc complexes: an odd evolutionary transition in a membrane protein structure

PubMed Central

Hasan, S. Saif; Cramer, William A.

2012-01-01

Lipid-binding sites and properties were compared in the hetero-oligomeric cytochrome (cyt) b6f and the yeast bc1 complexes that function, respectively, in photosynthetic and respiratory electron transport. Seven lipid-binding sites in the monomeric unit of the dimeric cyanobacterial b6f complex overlap four sites in the Chlamydomonas reinhardtii algal b6f complex and four in the yeast bc1 complex. The proposed lipid functions include: (i) interfacial–interhelix mediation between (a) the two 8-subunit monomers of the dimeric complex, (b) between the core domain (cyt b, subunit IV) and the six trans membrane helices of the peripheral domain (cyt f, iron–sulphur protein (ISP), and four small subunits in the boundary ‘picket fence’); (ii) stabilization of the ISP domain-swapped trans-membrane helix; (iii) neutralization of basic residues in the single helix of cyt f and of the ISP; (iv) a ‘latch’ to photosystem I provided by the β-carotene chain protruding through the ‘picket fence’; (v) presence of a lipid and chlorophyll a chlorin ring in b6f in place of the eighth helix in the bc1 cyt b polypeptide. The question is posed of the function of the lipid substitution in relation to the evolutionary change between the eight and seven helix structures of the cyt b polypeptide. On the basis of the known n-side activation of light harvesting complex II (LHCII) kinase by the p-side level of plastoquinol, one possibility is that the change was directed by the selective advantage of p- to n-side trans membrane signalling functions in b6f, with the lipid either mediating this function or substituting for the trans membrane helix of a signalling protein lost in crystallization. PMID:23148267

Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes

NASA Astrophysics Data System (ADS)

Miledi, Ricardo; Eusebi, Fabrizio; Martínez-Torres, Ataúlfo; Palma, Eleonora; Trettel, Flavia

2002-10-01

The Xenopus oocyte is a very powerful tool for studies of the structure and function of membrane proteins, e.g., messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional receptors and ion channels, and membrane vesicles from Torpedo electroplaques injected into oocytes fuse with the oocyte membrane and cause the appearance of functional Torpedo acetylcholine receptors and Cl channels. This approach was developed further to transplant already assembled neurotransmitter receptors from human brain cells to the plasma membrane of Xenopus oocytes. Membranes isolated from the temporal neocortex of a patient, operated for intractable epilepsy, were injected into oocytes and, within a few hours, the oocyte membrane acquired functional neurotransmitter receptors to -aminobutyric acid, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, kainate, and glycine. These receptors were also expressed in the plasma membrane of oocytes injected with mRNA extracted from the temporal neocortex of the same patient. All of this makes the Xenopus oocyte a more useful model than it already is for studies of the structure and function of many human membrane proteins and opens the way to novel pathophysiological investigations of some human brain disorders.

Insights into the structure and function of membrane-integrated processive glycosyltransferases

DOE PAGES

Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti; ...

2015-09-02

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. In this paper, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin,more » alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).« less

Insights into the structure and function of membrane-integrated processive glycosyltransferases

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

Bi, Yunchen; Hubbard, Caitlin; Purushotham, Pallinti

Complex carbohydrates perform essential functions in life, including energy storage, cell signaling, protein targeting, quality control, as well as supporting cell structure and stability. Extracellular polysaccharides (EPS) represent mainly structural polymers and are found in essentially all kingdoms of life. For example, EPS are important biofilm and capsule components in bacteria, represent major constituents in cell walls of fungi, algae, arthropods and plants, and modulate the extracellular matrix in vertebrates. Different mechanisms evolved by which EPS are synthesized. In this paper, we review the structures and functions of membrane-integrated processive glycosyltransferases (GTs) implicated in the synthesis and secretion of chitin,more » alginate, hyaluronan and poly-N-acetylglucosamine (PNAG).« less

Computer Modeling of Protocellular Functions: Peptide Insertion in Membranes

NASA Technical Reports Server (NTRS)

Rodriquez-Gomez, D.; Darve, E.; Pohorille, A.

2006-01-01

Lipid vesicles became the precursors to protocells by acquiring the capabilities needed to survive and reproduce. These include transport of ions, nutrients and waste products across cell walls and capture of energy and its conversion into a chemically usable form. In modem organisms these functions are carried out by membrane-bound proteins (about 30% of the genome codes for this kind of proteins). A number of properties of alpha-helical peptides suggest that their associations are excellent candidates for protobiological precursors of proteins. In particular, some simple a-helical peptides can aggregate spontaneously and form functional channels. This process can be described conceptually by a three-step thermodynamic cycle: 1 - folding of helices at the water-membrane interface, 2 - helix insertion into the lipid bilayer and 3 - specific interactions of these helices that result in functional tertiary structures. Although a crucial step, helix insertion has not been adequately studied because of the insolubility and aggregation of hydrophobic peptides. In this work, we use computer simulation methods (Molecular Dynamics) to characterize the energetics of helix insertion and we discuss its importance in an evolutionary context. Specifically, helices could self-assemble only if their interactions were sufficiently strong to compensate the unfavorable Free Energy of insertion of individual helices into membranes, providing a selection mechanism for protobiological evolution.

Bilayer Poly(Lactic-co-glycolic acid)/Nano-Hydroxyapatite Membrane with Barrier Function and Osteogenesis Promotion for Guided Bone Regeneration

PubMed Central

Fu, Li; Wang, Zhanfeng; Dong, Shujun; Cai, Yan; Ni, Yuxin; Zhang, Tianshou; Wang, Lin; Zhou, Yanmin

2017-01-01

Guided bone regeneration (GBR) is one such treatment that reconstructs neo-bone tissue by using a barrier membrane to prevent the invasion of soft tissue and to create a space for guiding new bone growth into the bone defect. Herein, we report a novel functionally graded bilayer membrane (FGBM) for GBR application. To fabricate the novel membrane, the composites of poly(lactic-co-glycolic acid) and nano-hydroxyapatite were prepared by phase inversion for the dense layer and by electrospinning for another porous layer, and their corresponding properties were evaluated including surface morphology, mechanics, degradability, cell barrier function, and in vitro osteogenic bioactivity. The results showed that PLGA with 5% nHA in dense layer could meet the requirement of mechanical strength and have excellent barrier function even on condition of post-degradation. Furthermore, PLGA with 30% nHA in porous layer could achieve the good physical and chemical properties. In addition, 30% nHA incorporation would enhance the in vitro mineralization, and have superior capabilities of cell adhesion, proliferation and differentiation compared to other groups. Therefore, the designed FGBM could potentially serve as a barrier for preferential tissue ingrowth and achieve a desirable therapeutic result for bone tissue regeneration. PMID:28772618

Transfer of metabolites across the peroxisomal membrane.

PubMed

Antonenkov, Vasily D; Hiltunen, J Kalervo

2012-09-01

Peroxisomes perform a large variety of metabolic functions that require a constant flow of metabolites across the membranes of these organelles. Over the last few years it has become clear that the transport machinery of the peroxisomal membrane is a unique biological entity since it includes nonselective channels conducting small solutes side by side with transporters for 'bulky' solutes such as ATP. Electrophysiological experiments revealed several channel-forming activities in preparations of plant, mammalian, and yeast peroxisomes and in glycosomes of Trypanosoma brucei. The properties of the first discovered peroxisomal membrane channel - mammalian Pxmp2 protein - have also been characterized. The channels are apparently involved in the formation of peroxisomal shuttle systems and in the transmembrane transfer of various water-soluble metabolites including products of peroxisomal β-oxidation. These products are processed by a large set of peroxisomal enzymes including carnitine acyltransferases, enzymes involved in the synthesis of ketone bodies, thioesterases, and others. This review discusses recent data pertaining to solute permeability and metabolite transport systems in peroxisomal membranes and also addresses mechanisms responsible for the transfer of ATP and cofactors such as an ATP transporter and nudix hydrolases. Copyright © 2012 Elsevier B.V. All rights reserved.

Long-Term Cognitive Outcome and Brain Imaging in Adults After Extracorporeal Membrane Oxygenation.

PubMed

von Bahr, Viktor; Kalzén, Håkan; Hultman, Jan; Frenckner, Björn; Andersson, Christin; Mosskin, Mikael; Eksborg, Staffan; Holzgraefe, Bernhard

2018-05-01

To investigate the presence of cognitive dysfunction and brain lesions in long-term survivors after treatment with extracorporeal membrane oxygenation for severe respiratory failure, and to see whether patients with prolonged hypoxemia were at increased risk. A single-center retrospective cohort study. Tertiary referral center for extracorporeal membrane oxygenation in Sweden. Long-term survivors treated between 1995 and July 2009. Seven patients from a previously published study investigated with a similar protocol were included. Brain imaging, neurocognitive testing, interview. Thirty-eight patients (i.e., n = 31 + 7) were enrolled and investigated in median 9.0 years after discharge. Only memory tests were performed in 10 patients, mainly due to a lack of formal education necessary for the test results to be reliable. Median full-scale intelligence quotient, memory index, and executive index were 97, 101, and 104, respectively (normal, 100 ± 15). Cognitive function was not reduced in the group with prolonged hypoxemia. Brain imaging showed cerebrovascular lesions in 14 of 38 patients (37%), most commonly in the group treated with venoarterial extracorporeal membrane oxygenation (7/11, 64%). In this group, memory function and executive function were significantly reduced. Patients treated with extracorporeal membrane oxygenation for respiratory failure may have normal cognitive function years after treatment, if not affected by cerebrovascular lesions. Permissive hypoxemia was not correlated with long-term cognitive dysfunction in the present study. Further prospective studies with minimal loss to follow-up are direly needed to confirm our findings.

Performance and Selectivity of Ceramic Membranes in the Ultrafiltration of Model Emulsion in Saline

NASA Astrophysics Data System (ADS)

Ćwirko, Konrad; Kalbarczyk-Jedynak, Agnieszka

2017-06-01

Oily wastewaters from different onshore and offshore installations and from maritime transport pose a serious threat to the environment so they must be treated by multistage separation also including membrane processes. The main advantages of such membranes are high performance and selectivity, high resistance for temperature and pressure, resistance for acids, bases and solvents, long service life and for application - significant reduction of industries and transport environmental impact. This work presents the results of the process of separation of oil from the emulsion with NaCl addition. Research was performed with a use of laboratory installation with ceramic 300 kDa membrane. The analysis concerned performance and selectivity of a membrane in the function of time and test results have been subsequently compared with the requirements of the IMO.

The role of phosphatidylinositol-transfer proteins at membrane contact sites.

PubMed

Selitrennik, Michael; Lev, Sima

2016-04-15

Phosphatidylinositol-transfer proteins (PITPs) have been initially identified as soluble factors that accelerate the monomeric exchange of either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayersin vitro They are highly conserved in eukaryotes and have been implicated in different cellular processes, including vesicular trafficking, signal transduction, and lipid metabolism. Recent studies suggest that PITPs function at membrane contact sites (MCSs) to facilitate the transport of PI from its synthesis site at the endoplasmic reticulum (ER) to various membrane compartments. In this review, we describe the underlying mechanism of PITPs targeting to MCSs, discuss their cellular roles and potential mode of action. © 2016 Authors; published by Portland Press Limited.

In-depth analysis of the thylakoid membrane proteome of Arabidopsis thaliana chloroplasts: new proteins, new functions, and a plastid proteome database.

PubMed

Friso, Giulia; Giacomelli, Lisa; Ytterberg, A Jimmy; Peltier, Jean-Benoit; Rudella, Andrea; Sun, Qi; Wijk, Klaas J van

2004-02-01

An extensive analysis of the Arabidopsis thaliana peripheral and integral thylakoid membrane proteome was performed by sequential extractions with salt, detergent, and organic solvents, followed by multidimensional protein separation steps (reverse-phase HPLC and one- and two-dimensional electrophoresis gels), different enzymatic and nonenzymatic protein cleavage techniques, mass spectrometry, and bioinformatics. Altogether, 154 proteins were identified, of which 76 (49%) were alpha-helical integral membrane proteins. Twenty-seven new proteins without known function but with predicted chloroplast transit peptides were identified, of which 17 (63%) are integral membrane proteins. These new proteins, likely important in thylakoid biogenesis, include two rubredoxins, a potential metallochaperone, and a new DnaJ-like protein. The data were integrated with our analysis of the lumenal-enriched proteome. We identified 83 out of 100 known proteins of the thylakoid localized photosynthetic apparatus, including several new paralogues and some 20 proteins involved in protein insertion, assembly, folding, or proteolysis. An additional 16 proteins are involved in translation, demonstrating that the thylakoid membrane surface is an important site for protein synthesis. The high coverage of the photosynthetic apparatus and the identification of known hydrophobic proteins with low expression levels, such as cpSecE, Ohp1, and Ohp2, indicate an excellent dynamic resolution of the analysis. The sequential extraction process proved very helpful to validate transmembrane prediction. Our data also were cross-correlated to chloroplast subproteome analyses by other laboratories. All data are deposited in a new curated plastid proteome database (PPDB) with multiple search functions (http://cbsusrv01.tc.cornell.edu/users/ppdb/). This PPDB will serve as an expandable resource for the plant community.

Functionally gradient material for membrane reactors to convert methane gas into value-added products

DOEpatents

Balachandran, U.; Dusek, J.T.; Kleefisch, M.S.; Kobylinski, T.P.

1996-11-12

A functionally gradient material for a membrane reactor for converting methane gas into value-added-products includes an outer tube of perovskite, which contacts air; an inner tube which contacts methane gas, of zirconium oxide, and a bonding layer between the perovskite and zirconium oxide layers. The bonding layer has one or more layers of a mixture of perovskite and zirconium oxide, with the layers transitioning from an excess of perovskite to an excess of zirconium oxide. The transition layers match thermal expansion coefficients and other physical properties between the two different materials. 7 figs.

Functionally gradient material for membrane reactors to convert methane gas into value-added products

DOEpatents

Balachandran, Uthamalingam; Dusek, Joseph T.; Kleefisch, Mark S.; Kobylinski, Thadeus P.

1996-01-01

A functionally gradient material for a membrane reactor for converting methane gas into value-added-products includes an outer tube of perovskite, which contacts air; an inner tube which contacts methane gas, of zirconium oxide, and a bonding layer between the perovskite and zirconium oxide layers. The bonding layer has one or more layers of a mixture of perovskite and zirconium oxide, with the layers transitioning from an excess of perovskite to an excess of zirconium oxide. The transition layers match thermal expansion coefficients and other physical properties between the two different materials.

Impulse control disorder, lysosomal malfunction and ATP13A2 insufficiency in Parkinsonism.

PubMed

Liu, Jun-Ping; Li, Jianfeng; Lu, Yanhua; Wang, Lihui; Chen, Gang

2017-02-01

Lysosomal transport of cargos in neurons is essential for neuronal proteostasis, transmission and functional motors and behaviours. Lysosomal malfunction including storage disorders is involved in the pathogenesis of Parkinson's disease (PD). Given the unclear molecular mechanisms of diverse defects in PD phenotypes, especially behavioural deficits, this mini review explores the cellular contexts of PD impulse control disorders and the molecular aspects of lysosomal cross-membrane transports. Focuses are paid to trace metal involvements in α-synuclein assembly in Lewy bodies, the functions and molecular interactions of ATP13A2 as ATPase transporters in lysosomal membranes for cross-membrane trafficking and lysosomal homeostasis, and our current understandings of the neural circuits in ICD. Erroneously polarized distributions of cargos such as metals and lipids on each side of lysosomal membranes triggered by gene mutations and deregulated expression of ATP13A2 may thus instigate sensing protein structural changes such as aggregations, organelle degeneration, and specific neuronal ageing and death in Parkinsonism. © 2016 John Wiley & Sons Australia, Ltd.

Nutrient Sensing at the Plasma Membrane of Fungal Cells.

PubMed

Van Dijck, Patrick; Brown, Neil Andrew; Goldman, Gustavo H; Rutherford, Julian; Xue, Chaoyang; Van Zeebroeck, Griet

2017-03-01

To respond to the changing environment, cells must be able to sense external conditions. This is important for many processes including growth, mating, the expression of virulence factors, and several other regulatory effects. Nutrient sensing at the plasma membrane is mediated by different classes of membrane proteins that activate downstream signaling pathways: nontransporting receptors, transceptors, classical and nonclassical G-protein-coupled receptors, and the newly defined extracellular mucin receptors. Nontransporting receptors have the same structure as transport proteins, but have lost the capacity to transport while gaining a receptor function. Transceptors are transporters that also function as a receptor, because they can rapidly activate downstream signaling pathways. In this review, we focus on these four types of fungal membrane proteins. We mainly discuss the sensing mechanisms relating to sugars, ammonium, and amino acids. Mechanisms for other nutrients, such as phosphate and sulfate, are discussed briefly. Because the model yeast Saccharomyces cerevisiae has been the most studied, especially regarding these nutrient-sensing systems, each subsection will commence with what is known in this species.

ER-plasma membrane junctions: Why and how do we study them?

PubMed

Chang, Chi-Lun; Chen, Yu-Ju; Liou, Jen

2017-09-01

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are membrane microdomains important for communication between the ER and the PM. ER-PM junctions were first reported in muscle cells in 1957, but mostly ignored in non-excitable cells due to their scarcity and lack of functional significance. In 2005, the discovery of stromal interaction molecule 1 (STIM1) mediating a universal Ca 2+ feedback mechanism at ER-PM junctions in mammalian cells led to a resurgence of research interests toward ER-PM junctions. In the past decade, several major advancements have been made in this emerging topic in cell biology, including the generation of tools for labeling ER-PM junctions and the unraveling of mechanisms underlying regulation and functions of ER-PM junctions. This review summarizes early studies, recently developed tools, and current advances in the characterization and understanding of ER-PM junctions. This article is part of a Special Issue entitled: Membrane Contact Sites edited by Christian Ungermann and Benoit Kornmann. Copyright © 2017 Elsevier B.V. All rights reserved.

Vertebrate Membrane Proteins: Structure, Function, and Insights from Biophysical Approaches

PubMed Central

MÜLLER, DANIEL J.; WU, NAN; PALCZEWSKI, KRZYSZTOF

2008-01-01

Membrane proteins are key targets for pharmacological intervention because they are vital for cellular function. Here, we analyze recent progress made in the understanding of the structure and function of membrane proteins with a focus on rhodopsin and development of atomic force microscopy techniques to study biological membranes. Membrane proteins are compartmentalized to carry out extra- and intracellular processes. Biological membranes are densely populated with membrane proteins that occupy approximately 50% of their volume. In most cases membranes contain lipid rafts, protein patches, or paracrystalline formations that lack the higher-order symmetry that would allow them to be characterized by diffraction methods. Despite many technical difficulties, several crystal structures of membrane proteins that illustrate their internal structural organization have been determined. Moreover, high-resolution atomic force microscopy, near-field scanning optical microscopy, and other lower resolution techniques have been used to investigate these structures. Single-molecule force spectroscopy tracks interactions that stabilize membrane proteins and those that switch their functional state; this spectroscopy can be applied to locate a ligand-binding site. Recent development of this technique also reveals the energy landscape of a membrane protein, defining its folding, reaction pathways, and kinetics. Future development and application of novel approaches during the coming years should provide even greater insights to the understanding of biological membrane organization and function. PMID:18321962

Layer-by-layer self-assembly in the development of electrochemical energy conversion and storage devices from fuel cells to supercapacitors.

PubMed

Xiang, Yan; Lu, Shanfu; Jiang, San Ping

2012-11-07

As one of the most effective synthesis tools, layer-by-layer (LbL) self-assembly technology can provide a strong non-covalent integration and accurate assembly between homo- or hetero-phase compounds or oppositely charged polyelectrolytes, resulting in highly-ordered nanoscale structures or patterns with excellent functionalities and activities. It has been widely used in the developments of novel materials and nanostructures or patterns from nanotechnologies to medical fields. However, the application of LbL self-assembly in the development of highly efficient electrocatalysts, specific functionalized membranes for proton exchange membrane fuel cells (PEMFCs) and electrode materials for supercapacitors is a relatively new phenomenon. In this review, the application of LbL self-assembly in the development and synthesis of key materials of PEMFCs including polyelectrolyte multilayered proton-exchange membranes, methanol-blocking Nafion membranes, highly uniform and efficient Pt-based electrocatalysts, self-assembled polyelectrolyte functionalized carbon nanotubes (CNTs) and graphenes will be reviewed. The application of LbL self-assembly for the development of multilayer nanostructured materials for use in electrochemical supercapacitors will also be reviewed and discussed (250 references).

Nanofiber Composite Membranes for Alkaline Fuel Cells: Generation of Compositional, Morphological, and Functional Property Relationships

DTIC Science & Technology

2015-12-01

and chemical stability. Also in Year 3, membranes were prepared by simultaneously electrospinning brominated poly(phenylene oxide) ( PPO ) and... PPO fibers (preventing water solubility when charged groups were added to the PPO ), as shown in Scheme 3. Subsequent mat processing included...brominated PPO fibers), and reaction of the resulting films with either trimethylamine or 1,2-dimethylimidazole to create cationic groups at those

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant

PubMed Central

Kim, Hak Jun; Lee, Jun Hyuck; Hur, Young Baek; Lee, Chang Woo; Park, Sun-Ha; Koo, Bon-Won

2017-01-01

Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis (TH), ice recrystallization inhibition (IRI), and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type. PMID:28134801

Membrane hydrophone phase characteristics through nonlinear acoustics measurements.

PubMed

Bloomfield, Philip E; Gandhi, Gaurav; Lewin, Peter A

2011-11-01

This work considers the need for both the amplitude and phase to fully characterize polyvinylidene fluoride (PVDF) membrane hydrophones and presents a comprehensive discussion of the nonlinear acoustic measurements utilized to extract the phase information and the experimental results taken with two widely used PVDF membrane hydrophones up to 100 MHz. A semi-empirical computer model utilized the hyperbolic propagation operator to predict the nonlinear pressure field and provide the complex frequency response of the corresponding source transducer. The PVDF hydrophone phase characteristics, which were obtained directly from the difference between the computer-modeled nonlinear field simulation and the corresponding measured harmonic frequency phase values, agree to within 10% with the phase predictions obtained from receive-transfer-function simulations based on software modeling of the membrane's physical properties. Cable loading effects and membrane hydrophone resonances were distinguished and identified through a series of impedance measurements and receive transfer function simulations on the hydrophones including their hard-wired coaxial cables. The results obtained indicate that the PVDF membrane hydrophone's phase versus frequency plot exhibits oscillations about a monotonically decreasing line. The maxima and minima inflection point slopes occur at the membrane thickness resonances and antiresonances, respectively. A cable resonance was seen at 100 MHz for the hydrophone with a 1-m cable attached, but not seen for the hydrophone with a shorter 0.65-m cable.

Manipulating lipid membrane architecture by liquid crystal-analog curvature elasticity (Presentation Recording)

NASA Astrophysics Data System (ADS)

Lee, Sin-Doo

2015-10-01

Soft matters such as liquid crystals and biological molecules exhibit a variety of interesting physical phenomena as well as new applications. Recently, in mimicking biological systems that have the ability to sense, regulate, grow, react, and regenerate in a highly responsive and self-adaptive manner, the significance of the liquid crystal order in living organisms, for example, a biological membrane possessing the lamellar order, is widely recognized from the viewpoints of physics and chemistry of interfaces and membrane biophysics. Lipid bilayers, resembling cell membranes, provide primary functions for the transport of biological components of ions and molecules in various cellular activities, including vesicle budding and membrane fusion, through lateral organization of the membrane components such as proteins. In this lecture, I will describe how the liquid crystal-analog curvature elasticity of a lipid bilayer plays a critical role in developing a new platform for understanding diverse biological functions at a cellular level. The key concept is to manipulate the local curvature at an interface between a solid substrate and a model membrane. Two representative examples will be demonstrated: one of them is the topographic control of lipid rafts in a combinatorial array where the ligand-receptor binding event occurs and the other concerns the reconstitution of a ring-type lipid raft in bud-mimicking architecture within the framework of the curvature elasticity.

Amino-Functionalized Ceramic Capillary Membranes for Controlled Virus Retention.

PubMed

Bartels, Julia; Souza, Marina N; Schaper, Amelie; Árki, Pál; Kroll, Stephen; Rezwan, Kurosch

2016-02-16

A straightforward chemical functionalization strategy using aminosilanes for high-flux yttria-stabilized zirconia capillary membranes is presented (macroporous, d50 = 144 nm, open porosity =49%, membrane flux ∼150 L/(m(2)hbar)). Three different aminosilanes with one, two or three amino groups per silane molecule, namely 3-aminopropyltriethoxysilane (APTES), N-(2-aminoethyl)-3-aminopropyltriethoxysilane (AE-APTES) and N-(3-trimethoxysilylpropyl)diethylenetriamine (TPDA), are used to generate the amino-functionalized membranes. With a higher number of amino groups per silane molecule increased loading capacities between 0.44 and 1.01 accessible amino groups/nm(2) membrane are achieved. Streaming potential measurements confirm that the zeta-potential of the membrane surface is converted from negative (non-functionalized) to positive (amino-functionalized). By operation in dead-end filtration mode using the model virus MS2 (diameter = 25 nm, IEP = 3.9) the virus retention capacity of the amino-functionalized membranes is significantly increased and log reduction values (LRVs) of up to 9.6 ± 0.3 (TPDA) are obtained whereas a LRV < 0.3 is provided by the non-functionalized membranes. Long-term dead-end filtration experiments for 1 week reveal a high stability of immobilized aminosilanes (TPDA), being robust against leaching. By iterative backflushing with desorption buffer MS2-loaded membranes are successfully regenerated being reusable for a new filtration cycle. The presented functionalization platform is highly promising for controlled virus retention.

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins.

PubMed

Sanjuán Szklarz, Luiza K; Kozjak-Pavlovic, Vera; Vögtle, F-Nora; Chacinska, Agnieszka; Milenkovic, Dusanka; Vogel, Sandra; Dürr, Mark; Westermann, Benedikt; Guiard, Bernard; Martinou, Jean-Claude; Borner, Christoph; Pfanner, Nikolaus; Meisinger, Chris

2007-04-20

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.

The Expanding Complexity of Estrogen Receptor Signaling in the Cardiovascular System

PubMed Central

Menazza, Sara; Murphy, Elizabeth

2016-01-01

Estrogen has important effects on cardiovascular function including regulation of vascular function, blood pressure, endothelial relaxation, the development of hypertrophy and cardioprotection. However, the mechanisms by which estrogen mediates these effects are still poorly understood. As detailed in this review, estrogen can regulate transcription by binding to two nuclear receptors, ERα and ERβ, which differentially regulate gene transcription. ERα and ERβ regulation of gene transcription is further modulated by tissue specific co-activators and co-repressors. Estrogen can bind to ERα and ERβ localized at the plasma membrane as well as GPER to initiate membrane delimited signaling, which enhances kinase signaling pathways that can have acute and long term effects. The kinase signaling pathways can also mediate transcriptional changes, and can synergize with the estrogen receptor to regulate cell function. This review will summarize the beneficial effects of estrogen in protecting the cardiovascular system through ER-dependent mechanisms with an emphasis on the role of the recently described ER-membrane signaling mechanisms. PMID:26838792

Biosynthesis and function of plant lipids

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

Thomson, W.W.; Mudd, J.B.; Gibbs, M.

The Sixth Annual Symposium in Botany and Plant Physiology was held January 13-15, 1983, at the University of California, Riverside. This volume comprises the papers that were presented. Subjects discussed at the symposium covered a wide range in the field of plant lipids. Biosynthesis of lipids occupied an important fraction of the presentations at the symposium. Subjects included detailed studies of the enzymes of fatty acid synthesis, several discussions of the incorporation of fatty acids into glycerolipids and the further modification of the fatty acids, and the synthesis of glycerolipids and desaturation of fatty acids in both maturing oilseeds andmore » chloroplasts. The physicochemical studies of glycerolipids and sterols in artificial membranes have led to distinct conclusions about their behaviour which must be relevant in the biological membrane. Results on the functional consequences of modifying the galactolipid composition in the chloroplast were an encouraging sign of progress in the attempts to relate membrane lipid composition to physiological function.« less

Protein quality control at the inner nuclear membrane

PubMed Central

Khmelinskii, Anton; Blaszczak, Ewa; Pantazopoulou, Marina; Fischer, Bernd; Omnus, Deike J.; Le Dez, Gaëlle; Brossard, Audrey; Gunnarsson, Alexander; Barry, Joseph D.; Meurer, Matthias; Kirrmaier, Daniel; Boone, Charles; Huber, Wolfgang; Rabut, Gwenaël; Ljungdahl, Per O.; Knop, Michael

2015-01-01

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum (ER) and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by ER-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc72,3. However, little is known regarding protein quality control at the INM. Here we describe a protein degradation pathway at the INM mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi34. We report that the As complex functions together with the ubiquitin conjugating enzymes Ubc6andUbc7to degrade soluble and integral membrane proteins. Genetic evidence suggest that the Asi ubiquitin ligase defines a pathway distinct from but complementary to ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer (tFT)5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquity ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalised integral membrane proteins, thus acting to maintain and safeguard the identity of the INM. PMID:25519137

Palisade is required in the Drosophila ovary for assembly and function of the protective vitelline membrane.

PubMed

Elalayli, Maggie; Hall, Jacklyn D; Fakhouri, Mazen; Neiswender, Hannah; Ellison, Tambrea T; Han, Zhe; Roon, Penny; LeMosy, Ellen K

2008-07-15

The innermost layer of the Drosophila eggshell, the vitelline membrane, provides structural support and positional information to the embryo. It is assembled in an incompletely understood manner from four major proteins to form a homogeneous, transparent extracellular matrix. Here we show that RNAi knockdown or genetic deletion of a minor constituent of this matrix, Palisade, results in structural disruptions during the initial synthesis of the vitelline membrane by somatic follicle cells surrounding the oocyte, including wide size variation among the precursor vitelline bodies and disorganization of follicle cell microvilli. Loss of Palisade or the microvillar protein Cad99C results in abnormal uptake into the oocyte of sV17, a major vitelline membrane protein, and defects in non-disulfide cross-linking of sV17 and sV23, while loss of Palisade has additional effects on processing and disulfide cross-linking of these proteins. Embryos surrounded by the abnormal vitelline membranes synthesized when Palisade is reduced are fertilized but undergo developmental arrest, usually during the first 13 nuclear divisions, with a nuclear phenotype of chromatin margination similar to that described for wild-type embryos subjected to anoxia. Our results demonstrate that Palisade is involved in coordinating assembly of the vitelline membrane and is required for functional properties of the eggshell.

Palisade is required in the Drosophila ovary for assembly and function of the protective vitelline membrane

PubMed Central

Elalayli, Maggie; Hall, Jacklyn D.; Fakhouri, Mazen; Neiswender, Hannah; Ellison, Tambrea T.; Han, Zhe; Roon, Penny; LeMosy, Ellen K.

2008-01-01

The innermost layer of the Drosophila eggshell, the vitelline membrane, provides structural support and positional information to the embryo. It is assembled in an incompletely understood manner from four major proteins to form a homogeneous, transparent extracellular matrix. Here we show that RNAi knockdown or genetic deletion of a minor constituent of this matrix, Palisade, results in structural disruptions during the initial synthesis of the vitelline membrane by somatic follicle cells surrounding the oocyte, including wide size variation among the precursor vitelline bodies and disorganization of follicle cell microvilli. Loss of Palisade or the microvillar protein Cad99C results in abnormal uptake into the oocyte of sV17, a major vitelline membrane protein, and defects in non-disulfide cross-linking of sV17 and sV23, while loss of Palisade has additional effects on processing and disulfide cross-linking of these proteins. Embryos surrounded by the abnormal vitelline membranes synthesized when Palisade is reduced are fertilized but undergo developmental arrest, usually during the first 13 nuclear divisions, with a nuclear phenotype of chromatin margination similar to that described for wild-type embryos subjected to anoxia. Our results demonstrate that Palisade is involved in coordinating assembly of the vitelline membrane and is required for functional properties of the eggshell. PMID:18514182

Function of plasma membrane microdomain-associated proteins during legume nodulation.

PubMed

Qiao, Zhenzhen; Libault, Marc

2017-10-03

Plasma membrane microdomains are plasma membrane sub-compartments enriched in sphingolipids and sterols, and composed by a specific set of proteins. They are involved in recognizing signal molecules, transducing these signals, and controlling endocytosis and exocytosis processes. In a recent study, applying biochemical and microscopic methods, we characterized the soybean GmFWL1 protein, a major regulator of soybean nodulation, as a new membrane microdomain-associated protein. Interestingly, upon rhizobia inoculation of the soybean root system, GmFWL1 and one of its interacting partners, GmFLOT2/4, both translocate to the root hair cell tip, the primary site of interaction and infection between soybean and Rhizobium. The role of GmFWL1 as a plasma membrane microdomain-associated protein is also supported by immunoprecipitation assays performed on soybean nodules, which revealed 178 GmFWL1 protein partners including a large number of microdomain-associated proteins such as GmFLOT2/4. In this addendum, we provide additional information about the identity of the soybean proteins repetitively identified as GmFWL1 protein partners. Their function is discussed especially in regard to plant-microbe interactions and microbial symbiosis. This addendum will provide new insights in the role of plasma membrane microdomains in regulating legume nodulation.

Elucidating the structure and function of S100 proteins in membranes

NASA Astrophysics Data System (ADS)

Valenzuela, Stella M.; Berkahn, Mark; Martin, Donald K.; Huynh, Thuan; Yang, Zheng; Geczy, Carolyn L.

2006-01-01

S100 proteins are important Ca 2+-binding proteins involved in vital cellular functions including the modulation of cell growth, migration and differentiation, regulation of intracellular signal transduction/phosphorylation pathways, energy metabolism, cytoskeletal interactions and modulation of ion channels. Furthermore, they are implicated in oncogenesis and numerous other disease states. Three S100 proteins: S100A8, S100A9 and S100A12 are constitutively expressed in neutrophils and monocytes. At low levels of intracellular Ca 2+, S100A8 and S100A9 are located predominantly in the cytosol but when Ca 2+ concentrations are elevated as a consequence of activation, they translocate to membranes and complex with cytoskeletal components such as vimentin. The functions of S100A8 and S100A9 at the plasma membrane remain unclear. A possible role may be the regulation of ion channel proteins. The current study uses the techniques of Atomic Force Microscopy and production of artificial lipid membranes in the form of liposomes to investigate possible mechanisms for the insertion of these proteins into membranes in order to elucidate their structure and stoichiometry in the transmembrane state. We have successfully imaged the liposomes as a lipid bilayer, the S100A8/A9 protein complex in solution and the S100A8/A9 complex associating with lipid, using tapping-mode atomic force microscopy, in buffer.

Shaping intercellular channels of plasmodesmata: the structure-to-function missing link.

PubMed

Nicolas, William J; Grison, Magali S; Bayer, Emmanuelle M

2017-12-18

Plasmodesmata (PD) are a hallmark of the plant kingdom and a cornerstone of plant biology and physiology, forming the conduits for the cell-to-cell transfer of proteins, RNA and various metabolites, including hormones. They connect the cytosols and endomembranes of cells, which allows enhanced cell-to-cell communication and synchronization. Because of their unique position as intercellular gateways, they are at the frontline of plant defence and signalling and constitute the battleground for virus replication and spreading. The membranous organization of PD is remarkable, where a tightly furled strand of endoplasmic reticulum comes into close apposition with the plasma membrane, the two connected by spoke-like elements. The role of these structural features is, to date, still not completely understood. Recent data on PD seem to point in an unexpected direction, establishing a close parallel between PD and membrane contact sites and defining plasmodesmal membranes as microdomains. However, the implications of this new viewpoint are not fully understood. Aided by available phylogenetic data, this review attempts to reassess the function of the different elements comprising the PD and the relevance of membrane lipid composition and biophysics in defining specialized microdomains of PD, critical for their function. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Biomimetic devices functionalized by membrane channel proteins

NASA Astrophysics Data System (ADS)

Schmidt, Jacob

2004-03-01

We are developing a new family of active materials which derive their functional properties from membrane proteins. These materials have two primary components: the proteins and the membranes themselves. I will discuss our recent work directed toward development of a generic platform for a "plug-and-play" philosophy of membrane protein engineering. By creating a stable biomimetic polymer membrane a single molecular monolayer thick, we will enable the exploitation of the function of any membrane protein, from pores and pumps to sensors and energy transducers. Our initial work has centered on the creation, study, and characterization of the biomimetic membranes. We are attempting to make large areas of membrane monolayers using Langmuir-Blodgett film formation as well as through arrays of microfabricated black lipid membrane-type septa. A number of techniques allow the insertion of protein into the membranes. As a benchmark, we have been employing a model system of voltage-gated pore proteins, which have electrically controllable porosities. I will report on the progress of this work, the characterization of the membranes, protein insertion processes, and the yield and functionality of the composite.

Concise Review: Fetal Membranes in Regenerative Medicine: New Tricks from an Old Dog?

PubMed Central

2017-01-01

Abstract The clinical application of the fetal membranes dates back to nearly a century. Their use has ranged from superficial skin dressings to surgical wound closure. The applications of the fetal membranes are constantly evolving, and key to this is the uncovering of multiple populations of stem and stem‐like cells, each with unique properties that can be exploited for regenerative medicine. In addition to pro‐angiogenic and immunomodulatory properties of the stem and stem‐like cells arising from the fetal membranes, the dehydrated and/or decellularized forms of the fetal membranes have been used to support the growth and function of other cells and tissues, including adipose‐derived mesenchymal stem cells. This concise review explores the biological origin of the fetal membranes, a history of their use in medicine, and recent developments in the use of fetal membranes and their derived stem and stem‐like cells in regenerative medicine. Stem Cells Translational Medicine 2017;6:1767–1776 PMID:28834402

Manipulating membrane lipid profiles to restore T-cell function in autoimmunity.

PubMed

Waddington, Kirsty E; Jury, Elizabeth C

2015-08-01

Plasma membrane lipid rafts are heterogeneous cholesterol and glycosphingolipid (GSL)-enriched microdomains, within which the tight packing of cholesterol with the saturated-acyl chains of GSLs creates a region of liquid-order relative to the surrounding disordered membrane. Thus lipid rafts govern the lateral mobility and interaction of membrane proteins and regulate a plethora of signal transduction events, including T-cell antigen receptor (TCR) signalling. The pathways regulating homoeostasis of membrane cholesterol and GSLs are tightly controlled and alteration of these metabolic processes coincides with immune cell dysfunction as is evident in atherosclerosis, cancer and autoimmunity. Indeed, membrane lipid composition is emerging as an important factor influencing the ability of cells to respond appropriately to microenvironmental stimuli. Consequently, there is increasing interest in targeting membrane lipids or their metabolic control as a novel therapeutic approach to modulate immune cell behaviour and our recent work demonstrates that this is a promising strategy in T-cells from patients with the autoimmune disease systemic lupus erythematosus (SLE). © 2015 Authors; published by Portland Press Limited.

Red cell membrane skeleton: structure-function relationships.

PubMed

Palek, J; Liu, S C

1980-01-01

This papaer reviews our present understanding of ultrastructure, organization, and functional characteristics of the erythrocyte membrane cytoskeleton. This two-dimensional fibrillar network of submembrane proteins can be visualized after extraction of lipids and integral membrane proteins by Triton X-100. Current data suggest that the major structural components of the cytoskeleton are heterodimers of double-stranded spectrin that form tetramers by head-to-head associations. The tetramers may be connected into a fibrillar meshwork by oligomers of actin. The control of membrane integrity by this network is illustrated by examples of two hemolyotic anemias characterized by marked membrane instability and vesiculation: 1) hereditary spherocytic anemia of the house mouse associated with spectrin deficiency and 2) hereditary pyropoikilocytosis, a hemolytic anemia in man characterized by thermal instability of the membrane and the presence of abnormal spectrin, which exhibits an increased propensity to thermal denaturation. Stabilization of the cytoskeletal network by covalent cross-links between the nearest cytoskeletal and integral membrane proteins results in a decrease of membrane deformability and a fixation of erythrocytes in their abnormal shape. Such cross-linkings include: 1) transamidative cross-links produced by introduction of Ca2+ (>0.5 mM) into fresh erythrocytes, and 2) intermolecular disulfide couplings, which are formed after extensive oxidation of fresh erythrocytes or after mild oxidation of ATP-depleted, but not fresh, erythrocytes. The significance of these cross-links in stabilization of shape of abnormal erythrocytes such as schistocytes remains to be determined. We conclude that spectrin and actin form a fibrillar submembrane network that plays an important role in control of membrane integrity, erythrocyte deformability, and stabilization of cells in abnormal shapes.

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components

PubMed Central

Pirbadian, Sahand; Barchinger, Sarah E.; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A.; Reed, Samantha B.; Romine, Margaret F.; Saffarini, Daad A.; Shi, Liang; Gorby, Yuri A.; Golbeck, John H.; El-Naggar, Mohamed Y.

2014-01-01

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic–abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution. PMID:25143589

Shewanella oneidensis MR-1 nanowires are outer membrane and periplasmic extensions of the extracellular electron transport components.

PubMed

Pirbadian, Sahand; Barchinger, Sarah E; Leung, Kar Man; Byun, Hye Suk; Jangir, Yamini; Bouhenni, Rachida A; Reed, Samantha B; Romine, Margaret F; Saffarini, Daad A; Shi, Liang; Gorby, Yuri A; Golbeck, John H; El-Naggar, Mohamed Y

2014-09-02

Bacterial nanowires offer an extracellular electron transport (EET) pathway for linking the respiratory chain of bacteria to external surfaces, including oxidized metals in the environment and engineered electrodes in renewable energy devices. Despite the global, environmental, and technological consequences of this biotic-abiotic interaction, the composition, physiological relevance, and electron transport mechanisms of bacterial nanowires remain unclear. We report, to our knowledge, the first in vivo observations of the formation and respiratory impact of nanowires in the model metal-reducing microbe Shewanella oneidensis MR-1. Live fluorescence measurements, immunolabeling, and quantitative gene expression analysis point to S. oneidensis MR-1 nanowires as extensions of the outer membrane and periplasm that include the multiheme cytochromes responsible for EET, rather than pilin-based structures as previously thought. These membrane extensions are associated with outer membrane vesicles, structures ubiquitous in Gram-negative bacteria, and are consistent with bacterial nanowires that mediate long-range EET by the previously proposed multistep redox hopping mechanism. Redox-functionalized membrane and vesicular extensions may represent a general microbial strategy for electron transport and energy distribution.

Preparation of proton conducting membranes containing bifunctional titania nanoparticles

NASA Astrophysics Data System (ADS)

Aslan, Ayşe; Bozkurt, Ayhan

2013-07-01

Throughout this work, the synthesis and characterization of novel proton conducting nanocomposite membranes including binary and ternary mixtures of sulfated nano-titania (TS), poly(vinyl alcohol) (PVA), and nitrilotri(methyl phosphonic acid) (NMPA) are discussed. The materials were produced by means of two different approaches where in the first, PVA and TS (10-15 nm) were admixed to form a binary system. The second method was the ternary nanocomposite membranes including PVA/TS/NMPA that were prepared at several compositions to get PVA-TS-(NMPA) x . The interaction of functional nano particles and NMPA in the host matrix was explored by FT-IR spectroscopy. The homogeneous distribution of bifunctional nanoparticles in the membrane was confirmed by SEM micrographs. The spectroscopic measurements and water/methanol uptake studies suggested a complexation between PVA and NMPA, which inhibited the leaching of the latter. The thermogravimetry analysis results verified that the presence of TS in the composite membranes suppressed the formation of phosphonic acid anhydrides up to 150 °C. The maximum proton conductivity has been measured for PVA-TS-(NMPA)3 as 0.003 S cm-1 at 150 °C.

Benchmark data for identifying multi-functional types of membrane proteins.

PubMed

Wan, Shibiao; Mak, Man-Wai; Kung, Sun-Yuan

2016-09-01

Identifying membrane proteins and their multi-functional types is an indispensable yet challenging topic in proteomics and bioinformatics. In this article, we provide data that are used for training and testing Mem-ADSVM (Wan et al., 2016. "Mem-ADSVM: a two-layer multi-label predictor for identifying multi-functional types of membrane proteins" [1]), a two-layer multi-label predictor for predicting multi-functional types of membrane proteins.

Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H+-translocating pyrophosphatases.

PubMed

Perez-Castineira, Jose R; Lopez-Marques, Rosa L; Villalba, Jose M; Losada, Manuel; Serrano, Aurelio

2002-12-10

Two types of proteins that hydrolyze inorganic pyrophosphate (PPi), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping pyrophosphatases (sPPases and H(+)-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PPi releasing heat, whereas H+-PPases, so far unidentified in animal and fungal cells, couple the energy of PPi hydrolysis to proton movement across biological membranes. The budding yeast Saccharomyces cerevisiae has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (IPP1) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional S. cerevisiae mutant (named YPC-1) whose functional IPP1 gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H+-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H+-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PPi is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H+-PPases can mediate net hydrolysis of PPi in vivo. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure-function relationships of this type of proton pumps.

Preparation of Gap Junctions in Membrane Microdomains for Immunoprecipitation and Mass Spectrometry Interactome Analysis.

PubMed

Fowler, Stephanie; Akins, Mark; Bennett, Steffany A L

2016-01-01

Protein interaction networks at gap junction plaques are increasingly implicated in a variety of intracellular signaling cascades. Identifying protein interactions of integral membrane proteins is a valuable tool for determining channel function. However, several technical challenges exist. Subcellular fractionation of the bait protein matrix is usually required to identify less abundant proteins in complex homogenates. Sufficient solvation of the lipid environment without perturbation of the protein interactome must also be achieved. The present chapter describes the flotation of light and heavy liver tissue membrane microdomains to facilitate the identification and analysis of endogenous gap junction proteins and includes technical notes for translation to other integral membrane proteins, tissues, or cell culture models. These procedures are valuable tools for the enrichment of gap junction membrane compartments and for the identification of gap junction signaling interactomes.

[Application of the xenogenic acellular dermal matrix membrane application used in the postoperative tissue shortage repair].

PubMed

Bai, Yanxia; Yan, Liying; Zhang, Shaoqiang; Shao, Yuan; Yao, Xiaobao; Li, Honghui; Zhao, Ruimin; Zhao, Qian; Zhang, Pengfei; Yang, Qi

2014-09-01

To observe the short-term and long-term curative effect of the xenogenic acellular dermal matrix membrane (or joint muscle flap transfer) application used in the 82 cases postoperative tissue shortage repair that after the head neck carcinoma resection. To held the 82 cases head neck carcinoma postoperative mucosa shortage repaired after resection by the xenogenic acellular dermal matrix membrane (or joint muscle flap transfer), 65 cases mucosa shortage wound be directly covered by the repair membrane and the other 17 cases mucosa shortage wound be repaired by the tranfered muscle tissue flap with the repair membrane covered; 53 cases underwent additional postoperative radiotherapy between 2-4 weeks and follow-up in 1, 3, 6, 12, 18, 24, 30, 36, 48, 60 months and observed the operation site repair process through the electronic laryngoscope, observed the patients respiration, swallow, phonation function. Seventy-seven cases patients operation incision reached I phase healing standard, another 5 cases patients operation incision reached II phase healing standard because of the wound infection and fully-recovered through the local wound drainage,dressing process. All the patients tracheal cannula,the stomach tube be extubated successfully and without the local cicatricial constriction occurred. Seventy-eight cases follow up period reached 1 year including 53 cases who underwent postoperative radiotherapy, 49 cases follow up period reached 3 years including 32 cases who underwent postoperative radiotherapy, 14 cases follow up period reached 5 years including 12 cases who underwent postoperative radiotherapy. The patients with static local lesions discovered no reaction such as exclusion, allergy. The application of xenogenic acellular dermal matrix membrane (or joint muscle flap transfer used in in the postoperative tissue shortage repair that after the head neck carcinoma resection have several advantage such as comparatively easily implementation, operation safety edge enough,well preserved organ function, comparatively low incidence about the laryngeal stenosis, the short-term and long-term repair effect are all exact.

The use of fibrous, supramolecular membranes and human tubular cells for renal epithelial tissue engineering: towards a suitable membrane for a bioartificial kidney.

PubMed

Dankers, Patricia Y W; Boomker, Jasper M; Huizinga-van der Vlag, Ali; Smedts, Frank M M; Harmsen, Martin C; van Luyn, Marja J A

2010-11-10

A bioartificial kidney, which is composed of a membrane cartridge with renal epithelial cells, can substitute important kidney functions in patients with renal failure. A particular challenge is the maintenance of monolayer integrity and specialized renal epithelial cell functions ex vivo. We hypothesized that this can be improved by electro-spun, supramolecular polymer membranes which show clear benefits in ease of processability. We found that after 7 d, in comparison to conventional microporous membranes, renal tubular cells cultured on top of our fibrous supramolecular membranes formed polarized monolayers, which is prerequisite for a well-functioning bioartificial kidney. In future, these supramolecular membranes allow for incorporation of peptides that may increase cell function even further.

The G protein alpha subunit (GP alpha1) is associated with the ER and the plasma membrane in meristematic cells of Arabidopsis and cauliflower.

PubMed

Weiss, C A; White, E; Huang, H; Ma, H

1997-05-05

Towards the elucidation of the cellular function(s) of GP alpha1, we have characterized its subcellular localization using immunofluorescence and cell fractionation. GP alpha1 is not present in nuclei or chloroplasts. It is a membrane-bound protein, and analysis of isolated endoplasmic and plasma membranes indicates a good correlation between GP alpha1 in both the plasma membrane and the ER compartment. Interestingly, these results may suggest more different functions for GP alpha1: it might be involved in transmission of extracellular signals across the plasma membrane and in the cytoplasm, and/or it may also be involved in regulating some aspects of the ER functions or membrane trafficking between both membranes.

Cellular Engineering with Membrane Fusogenic Liposomes to Produce Functionalized Extracellular Vesicles.

PubMed

Lee, Junsung; Lee, Hyoungjin; Goh, Unbyeol; Kim, Jiyoung; Jeong, Moonkyoung; Lee, Jean; Park, Ji-Ho

2016-03-23

Engineering of extracellular vesicles (EVs) without affecting biological functions remains a challenge, limiting the broad applications of EVs in biomedicine. Here, we report a method to equip EVs with various functional agents, including fluorophores, drugs, lipids, and bio-orthogonal chemicals, in an efficient and controlled manner by engineering parental cells with membrane fusogenic liposomes, while keeping the EVs intact. As a demonstration of how this method can be applied, we prepared EVs containing azide-lipids, and conjugated them with targeting peptides using copper-free click chemistry to enhance targeting efficacy to cancer cells. We believe that this liposome-based cellular engineering method will find utility in studying the biological roles of EVs and delivering therapeutic agents through their innate pathway.

Cell Membrane Coating Nanotechnology.

PubMed

Fang, Ronnie H; Kroll, Ashley V; Gao, Weiwei; Zhang, Liangfang

2018-06-01

Nanoparticle-based therapeutic, prevention, and detection modalities have the potential to greatly impact how diseases are diagnosed and managed in the clinic. With the wide range of nanomaterials available, the rational design of nanocarriers on an application-specific basis has become increasingly commonplace. Here, a comprehensive overview is provided on an emerging platform: cell-membrane-coating nanotechnology. As a fundamental unit of biology, cells carry out a wide range of functions, including the remarkable ability to interface and interact with their surrounding environment. Instead of attempting to replicate such functions via synthetic techniques, researchers are now directly leveraging naturally derived cell membranes as a means of bestowing nanoparticles with enhanced biointerfacing capabilities. This top-down technique is facile, highly generalizable, and has the potential to greatly augment existing nanocarriers. Further, the introduction of a natural membrane substrate onto nanoparticles surfaces has enabled additional applications beyond those traditionally associated with nanomedicine. Despite its relative youth, there exists an impressive body of literature on cell membrane coating, which is covered here in detail. Overall, there is still significant room for development, as researchers continue to refine existing workflows while finding new and exciting applications that can take advantage of this developing technology. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

PubMed

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

2015-12-10

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

Sterol Regulation of Voltage-Gated K+ Channels.

PubMed

Balajthy, Andras; Hajdu, Peter; Panyi, Gyorgy; Varga, Zoltan

2017-01-01

Cholesterol is an essential lipid building block of the cellular plasma membrane. In addition to its structural role, it regulates the fluidity and raft structure of the membrane and influences the course of numerous membrane-linked signaling pathways and the function of transmembrane proteins, including ion channels. This is supported by a vast body of scientific data, which demonstrates the modulation of ion channels with a great variety of ion selectivity, gating, and tissue distribution by changes in membrane cholesterol. Here, we review what is currently known about the modulation of voltage-gated K + (Kv) channels by changes in membrane cholesterol content, considering raft association of the channels, the roles of cholesterol recognition sites, and those of adaptor proteins in cholesterol-Kv channel interactions. We specifically focus on Kv1.3, the dominant K + channel of human T cells. Effects of cholesterol depletion and enrichment and 7-dehydrocholesterol enrichment on Kv1.3 gating are discussed in the context of the immunological synapse and the comparison of the in vitro effects of sterol modifications on Kv1.3 function with ex vivo effects on cells from hypercholesterolemic and Smith-Lemli-Opitz patients. © 2017 Elsevier Inc. All rights reserved.

Membrane fusion and exocytosis.

PubMed

Jahn, R; Südhof, T C

1999-01-01

Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

Nanodisc-Tm: Rapid functional assessment of nanodisc reconstituted membrane proteins by CPM assay.

PubMed

Ashok, Yashwanth; Jaakola, Veli-Pekka

2016-01-01

Membrane proteins are generally unstable in detergents. Therefore, biochemical and biophysical studies of membrane proteins in lipidic environments provides a near native-like environment suitable for membrane proteins. However, manipulation of proteins embedded in lipid bilayer has remained difficult. Methods such as nanodiscs and lipid cubic phase have been developed for easy manipulation of membrane proteins and have yielded significant insights into membrane proteins. Traditionally functional reconstitution of receptors in nanodiscs has been studied with radioligands. We present a simple and faster method for studying the functionality of reconstituted membrane proteins for routine characterization of protein batches after initial optimization of suitable conditions using radioligands. The benefits of the method are •Faster and generic method to assess functional reconstitution of membrane proteins.•Adaptable in high throughput format (≥96 well format).•Stability measurement in near-native lipid environment and lipid dependent melting temperatures.

Covalent enzyme immobilization onto carbon nanotubes using a membrane reactor

NASA Astrophysics Data System (ADS)

Voicu, Stefan Ioan; Nechifor, Aurelia Cristina; Gales, Ovidiu; Nechifor, Gheorghe

2011-05-01

Composite porous polysulfone-carbon nanotubes membranes were prepared by dispersing carbon nanotubes into a polysulfone solution followed by the membrane formation by phase inversion-immersion precipitation technique. The carbon nanotubes with amino groups on surface were functionalized with different enzymes (carbonic anhydrase, invertase, diastase) using cyanuric chloride as linker between enzyme and carbon nanotube. The composite membrane was used as a membrane reactor for a better dispersion of carbon nanotubes and access to reaction centers. The membrane also facilitates the transport of enzymes to active carbon nanotubes centers for functionalization (amino groups). The functionalized carbon nanotubes are isolated by dissolving the membranes after the end of reaction. Carbon nanotubes with covalent immobilized enzymes are used for biosensors fabrications. The obtained membranes were characterized by Scanning Electron Microscopy, Thermal analysis, FT-IR Spectroscopy, Nuclear Magnetic Resonance, and functionalized carbon nanotubes were characterized by FT-IR spectroscopy.

Examining hemodialyzer membrane performance using proteomic technologies

PubMed Central

Bonomini, Mario; Pieroni, Luisa; Di Liberato, Lorenzo; Sirolli, Vittorio; Urbani, Andrea

2018-01-01

The success and the quality of hemodialysis therapy are mainly related to both clearance and biocompatibility properties of the artificial membrane packed in the hemodialyzer. Performance of a membrane is strongly influenced by its interaction with the plasma protein repertoire during the extracorporeal procedure. Recognition that a number of medium–high molecular weight solutes, including proteins and protein-bound molecules, are potentially toxic has prompted the development of more permeable membranes. Such membrane engineering, however, may cause loss of vital proteins, with membrane removal being nonspecific. In addition, plasma proteins can be adsorbed onto the membrane surface upon blood contact during dialysis. Adsorption can contribute to the removal of toxic compounds and governs the biocompatibility of a membrane, since surface-adsorbed proteins may trigger a variety of biologic blood pathways with pathophysiologic consequences. Over the last years, use of proteomic approaches has allowed polypeptide spectrum involved in the process of hemodialysis, a key issue previously hampered by lack of suitable technology, to be assessed in an unbiased manner and in its full complexity. Proteomics has been successfully applied to identify and quantify proteins in complex mixtures such as dialysis outflow fluid and fluid desorbed from dialysis membrane containing adsorbed proteins. The identified proteins can also be characterized by their involvement in metabolic and signaling pathways, molecular networks, and biologic processes through application of bioinformatics tools. Proteomics may thus provide an actual functional definition as to the effect of a membrane material on plasma proteins during hemodialysis. Here, we review the results of proteomic studies on the performance of hemodialysis membranes, as evaluated in terms of solute removal efficiency and blood–membrane interactions. The evidence collected indicates that the information provided by proteomic investigations yields improved molecular and functional knowledge and may lead to the development of more efficient membranes for the potential benefit of the patient. PMID:29296087

Plasma membrane organization and dynamics is probe and cell line dependent.

PubMed

Huang, Shuangru; Lim, Shi Ying; Gupta, Anjali; Bag, Nirmalya; Wohland, Thorsten

2017-09-01

The action and interaction of membrane receptor proteins take place within the plasma membrane. The plasma membrane, however, is not a passive matrix. It rather takes an active role and regulates receptor distribution and function by its composition and the interaction of its lipid components with embedded and surrounding proteins. Furthermore, it is not a homogenous fluid but contains lipid and protein domains of various sizes and characteristic lifetimes which are important in regulating receptor function and signaling. The precise lateral organization of the plasma membrane, the differences between the inner and outer leaflet, and the influence of the cytoskeleton are still debated. Furthermore, there is a lack of comparisons of the organization and dynamics of the plasma membrane of different cell types. Therefore, we used four different specific membrane markers to test the lateral organization, the differences between the inner and outer membrane leaflet, and the influence of the cytoskeleton of up to five different cell lines, including Chinese hamster ovary (CHO-K1), Human cervical carcinoma (HeLa), neuroblastoma (SH-SY5Y), fibroblast (WI-38) and rat basophilic leukemia (RBL-2H3) cells by Imaging Total Internal Reflection (ITIR)-Fluorescence Correlation Spectroscopy (FCS). We measure diffusion in the temperature range of 298-310K to measure the Arrhenius activation energy (E Arr ) of diffusion and apply the FCS diffusion law to obtain information on the spatial organization of the probe molecules on the various cell membranes. Our results show clear differences of the FCS diffusion law and E Arr for the different probes in dependence of their localization. These differences are similar in the outer and inner leaflet of the membrane. However, these values can differ significantly between different cell lines raising the question how molecular plasma membrane events measured in different cell lines can be compared. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

Examining hemodialyzer membrane performance using proteomic technologies.

PubMed

Bonomini, Mario; Pieroni, Luisa; Di Liberato, Lorenzo; Sirolli, Vittorio; Urbani, Andrea

2018-01-01

The success and the quality of hemodialysis therapy are mainly related to both clearance and biocompatibility properties of the artificial membrane packed in the hemodialyzer. Performance of a membrane is strongly influenced by its interaction with the plasma protein repertoire during the extracorporeal procedure. Recognition that a number of medium-high molecular weight solutes, including proteins and protein-bound molecules, are potentially toxic has prompted the development of more permeable membranes. Such membrane engineering, however, may cause loss of vital proteins, with membrane removal being nonspecific. In addition, plasma proteins can be adsorbed onto the membrane surface upon blood contact during dialysis. Adsorption can contribute to the removal of toxic compounds and governs the biocompatibility of a membrane, since surface-adsorbed proteins may trigger a variety of biologic blood pathways with pathophysiologic consequences. Over the last years, use of proteomic approaches has allowed polypeptide spectrum involved in the process of hemodialysis, a key issue previously hampered by lack of suitable technology, to be assessed in an unbiased manner and in its full complexity. Proteomics has been successfully applied to identify and quantify proteins in complex mixtures such as dialysis outflow fluid and fluid desorbed from dialysis membrane containing adsorbed proteins. The identified proteins can also be characterized by their involvement in metabolic and signaling pathways, molecular networks, and biologic processes through application of bioinformatics tools. Proteomics may thus provide an actual functional definition as to the effect of a membrane material on plasma proteins during hemodialysis. Here, we review the results of proteomic studies on the performance of hemodialysis membranes, as evaluated in terms of solute removal efficiency and blood-membrane interactions. The evidence collected indicates that the information provided by proteomic investigations yields improved molecular and functional knowledge and may lead to the development of more efficient membranes for the potential benefit of the patient.

Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

PubMed Central

Owen, Caroline A.

2008-01-01

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: 1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinase by cells; 2) the availability of surface binding sites for proteinases; and/or 3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: 1) concentrating the activity of proteinases to the immediate pericellular environment; 2) facilitating pro-enzyme activation; 3) increasing proteinase stability and retention in the extracellular space; 4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and 5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes. PMID:18329945

Graphene membranes with nanoslits for seawater desalination via forward osmosis.

PubMed

Dahanayaka, Madhavi; Liu, Bo; Hu, Zhongqiao; Pei, Qing-Xiang; Chen, Zhong; Law, Adrian Wing-Keung; Zhou, Kun

2017-11-22

Stacked graphene (GE) membranes with cascading nanoslits can be synthesized economically compared to monolayer nanoporous GE membranes, and have potential for molecular separation. This study focuses on investigating the seawater desalination performance of these stacked GE layers as forward osmosis (FO) membranes by using molecular dynamics simulations. The FO performance is evaluated in terms of water flux and salt rejection and is explained by analysing the water density distribution and radial distribution function. The water flow displays an Arrhenius type relation with temperature and the activation energy for the stacked GE membrane is estimated to be 8.02 kJ mol -1 , a value much lower than that of commercially available FO membranes. The study reveals that the membrane characteristics including the pore width, offset, interlayer separation distance and number of layers have significant effects on the desalination performance. Unlike monolayer nanoporous GE membranes, at an optimum layer separation distance, the stacked GE membranes with large pore widths and completely misaligned pore configuration can retain complete ion rejection and maintain a high water flux. Findings from the present study are helpful in developing GE-based membranes for seawater desalination via FO.

Membrane Interactions of Phytochemicals as Their Molecular Mechanism Applicable to the Discovery of Drug Leads from Plants.

PubMed

Tsuchiya, Hironori

2015-10-16

In addition to interacting with functional proteins such as receptors, ion channels, and enzymes, a variety of drugs mechanistically act on membrane lipids to change the physicochemical properties of biomembranes as reported for anesthetic, adrenergic, cholinergic, non-steroidal anti-inflammatory, analgesic, antitumor, antiplatelet, antimicrobial, and antioxidant drugs. As well as these membrane-acting drugs, bioactive plant components, phytochemicals, with amphiphilic or hydrophobic structures, are presumed to interact with biological membranes and biomimetic membranes prepared with phospholipids and cholesterol, resulting in the modification of membrane fluidity, microviscosity, order, elasticity, and permeability with the potencies being consistent with their pharmacological effects. A novel mechanistic point of view of phytochemicals would lead to a better understanding of their bioactivities, an insight into their medicinal benefits, and a strategic implication for discovering drug leads from plants. This article reviews the membrane interactions of different classes of phytochemicals by highlighting their induced changes in membrane property. The phytochemicals to be reviewed include membrane-interactive flavonoids, terpenoids, stilbenoids, capsaicinoids, phloroglucinols, naphthodianthrones, organosulfur compounds, alkaloids, anthraquinonoids, ginsenosides, pentacyclic triterpene acids, and curcuminoids. The membrane interaction's applicability to the discovery of phytochemical drug leads is also discussed while referring to previous screening and isolating studies.

Reprogramming cellular functions with engineered membrane proteins.

PubMed

Arber, Caroline; Young, Melvin; Barth, Patrick

2017-10-01

Taking inspiration from Nature, synthetic biology utilizes and modifies biological components to expand the range of biological functions for engineering new practical devices and therapeutics. While early breakthroughs mainly concerned the design of gene circuits, recent efforts have focused on engineering signaling pathways to reprogram cellular functions. Since signal transduction across cell membranes initiates and controls intracellular signaling, membrane receptors have been targeted by diverse protein engineering approaches despite limited mechanistic understanding of their function. The modular architecture of several receptor families has enabled the empirical construction of chimeric receptors combining domains from distinct native receptors which have found successful immunotherapeutic applications. Meanwhile, progress in membrane protein structure determination, computational modeling and rational design promise to foster the engineering of a broader range of membrane receptor functions. Marrying empirical and rational membrane protein engineering approaches should enable the reprogramming of cells with widely diverse fine-tuned functions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Do inositol supplements enhance phosphatidylinositol supply and thus support endoplasmic reticulum function?

PubMed

Michell, Robert H

2018-06-03

This review attempts to explain why consuming extra myoinositol (Ins), an essential component of membrane phospholipids, is often beneficial for patients with conditions characterised by insulin resistance, non-alcoholic fatty liver disease and endoplasmic reticulum (ER) stress. For decades we assumed that most human diets provide an adequate Ins supply, but newer evidence suggests that increasing Ins intake ameliorates several disorders, including polycystic ovary syndrome, gestational diabetes, metabolic syndrome, poor sperm development and retinopathy of prematurity. Proposed explanations often suggest functional enhancement of minor facets of Ins Biology such as insulin signalling through putative inositol-containing 'mediators', but offer no explanation for this selectivity. It is more likely that eating extra Ins corrects a deficiency of an abundant Ins-containing cell constituent, probably phosphatidylinositol (PtdIns). Much of a cell's PtdIns is in ER membranes, and an increase in ER membrane synthesis, enhancing the ER's functional capacity, is often an important part of cell responses to ER stress. This review: (a) reinterprets historical information on Ins deficiency as describing a set of events involving a failure of cells adequately to adapt to ER stress; (b) proposes that in the conditions that respond to dietary Ins there is an overstretching of Ins reserves that limits the stressed ER's ability to make the 'extra' PtdIns needed for ER membrane expansion; and (c) suggests that eating Ins supplements increases the Ins supply to Ins-deficient and ER-stressed cells, allowing them to make more PtdIns and to expand the ER membrane system and sustain ER functions.

Electrochemical oxygen concentrator as an oxygen compressor

NASA Technical Reports Server (NTRS)

1975-01-01

A solid polymer electrolyte (SPE) oxygen compressor is described which generates pressures of 3000 psi. The SPE is a cation exchange membrane with chemical compatibility, and has the capability of withstanding 5000 psi. Other features of the compressor described include: gasketless sealing, porus plate cell supports, and conductive cooling. Results are presented of a computer program which defines the power of the system as a function of density, temperature, pressure, membrane thickness, and water content.

Impact of Lipid Composition and Receptor Conformation on the Spatio-temporal Organization of μ-Opioid Receptors in a Multi-component Plasma Membrane Model

PubMed Central

Marino, Kristen A.; Prada-Gracia, Diego; Provasi, Davide; Filizola, Marta

2016-01-01

The lipid composition of cell membranes has increasingly been recognized as playing an important role in the function of various membrane proteins, including G Protein-Coupled Receptors (GPCRs). For instance, experimental and computational evidence has pointed to lipids influencing receptor oligomerization directly, by physically interacting with the receptor, and/or indirectly, by altering the bulk properties of the membrane. While the exact role of oligomerization in the function of class A GPCRs such as the μ-opioid receptor (MOR) is still unclear, insight as to how these receptors oligomerize and the relevance of the lipid environment to this phenomenon is crucial to our understanding of receptor function. To examine the effect of lipids and different MOR conformations on receptor oligomerization we carried out extensive coarse-grained molecular dynamics simulations of crystal structures of inactive and/or activated MOR embedded in an idealized mammalian plasma membrane composed of 63 lipid types asymmetrically distributed across the two leaflets. The results of these simulations point, for the first time, to specific direct and indirect effects of the lipids, as well as the receptor conformation, on the spatio-temporal organization of MOR in the plasma membrane. While sphingomyelin-rich, high-order lipid regions near certain transmembrane (TM) helices of MOR induce an effective long-range attractive force on individual protomers, both long-range lipid order and interface formation are found to be conformation dependent, with a larger number of different interfaces formed by inactive MOR compared to active MOR. PMID:27959924

Engineering membrane scaffolds with both physical and biomolecular signaling.

PubMed

Tejeda-Montes, Esther; Smith, Katherine H; Poch, Marta; López-Bosque, María Jesús; Martín, Laura; Alonso, Matilde; Engel, Elisabeth; Mata, Alvaro

2012-03-01

We report on the combination of a top-down and bottom-up approach to develop thin bioactive membrane scaffolds based on functional elastin-like polymers (ELPs). Our strategy combines ELP cross-linking and assembly, and a variety of standard and novel micro/nanofabrication techniques to create self-supporting membranes down to ∼500 nm thick that incorporate both physical and biomolecular signals, which can be easily tailored for a specific application. In this study we used an ELP that included the cell-binding motif arginine-glycine-aspartic acid-serine (RGDS). Furthermore, fabrication processes were developed to create membranes that exhibited topographical patterns with features down to 200 nm in lateral dimensions and up to 10 μm in height on either one or both sides, uniform and well-defined pores, or multiple ELP layers. A variety of processing parameters were tested in order to optimize membrane fabrication, including ELP and cross-linker concentration, temperature, reaction time and ambient humidity. Membrane micro/nanopatterning, swelling and stiffness were characterized by atomic force microscopy, nanoindentation tests and scanning electron microscopy. Upon immersion in phosphate-buffered saline and an increase in temperature from 25 to 40°C, membranes exhibited a significant increase in surface stiffness, with the reduced Young's modulus increasing with temperature. Finally, rat mesenchymal stem cells were cultured on thin RGDS-containing membranes, which allowed cell adhesion, qualitatively enhanced spreading compared to membranes without RGDS epitopes and permitted proliferation. Furthermore, cell morphology was drastically affected by topographical patterns on the surface of the membranes. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Mechanosensation in an adipose fin

PubMed Central

2016-01-01

Adipose fins are found on approximately 20% of ray-finned fish species. The apparently rudimentary anatomy of adipose fins inspired a longstanding hypothesis that these fins are vestigial and lack function. However, adipose fins have evolved repeatedly within Teleostei, suggesting adaptive function. Recently, adipose fins were proposed to function as mechanosensors, detecting fluid flow anterior to the caudal fin. Here we test the hypothesis that adipose fins are mechanosensitive in the catfish Corydoras aeneus. Neural activity, recorded from nerves that innervate the fin, was shown to encode information on both movement and position of the fin membrane, including the magnitude of fin membrane displacement. Thus, the adipose fin of C. aeneus is mechanosensitive and has the capacity to function as a ‘precaudal flow sensor’. These data force re-evaluation of adipose fin clipping, a common strategy for tagging fishes, and inform hypotheses of how function evolves in novel vertebrate appendages. PMID:26984621

Mechanosensation in an adipose fin.

PubMed

Aiello, Brett R; Stewart, Thomas A; Hale, Melina E

2016-03-16

Adipose fins are found on approximately 20% of ray-finned fish species. The apparently rudimentary anatomy of adipose fins inspired a longstanding hypothesis that these fins are vestigial and lack function. However, adipose fins have evolved repeatedly within Teleostei, suggesting adaptive function. Recently, adipose fins were proposed to function as mechanosensors, detecting fluid flow anterior to the caudal fin. Here we test the hypothesis that adipose fins are mechanosensitive in the catfish Corydoras aeneus. Neural activity, recorded from nerves that innervate the fin, was shown to encode information on both movement and position of the fin membrane, including the magnitude of fin membrane displacement. Thus, the adipose fin of C. aeneus is mechanosensitive and has the capacity to function as a 'precaudal flow sensor'. These data force re-evaluation of adipose fin clipping, a common strategy for tagging fishes, and inform hypotheses of how function evolves in novel vertebrate appendages. © 2016 The Author(s).

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors*

PubMed Central

Stahlschmidt, Wiebke; Robertson, Mark J.; Robinson, Phillip J.; McCluskey, Adam; Haucke, Volker

2014-01-01

Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane. PMID:24407285

Quantification of arrestin-rhodopsin binding stoichiometry.

PubMed

Lally, Ciara C M; Sommer, Martha E

2015-01-01

We have developed several methods to quantify arrestin-1 binding to rhodopsin in the native rod disk membrane. These methods can be applied to study arrestin interactions with all functional forms of rhodopsin, including dark-state rhodopsin, light-activated metarhodopsin II (Meta II), and the products of Meta II decay, opsin and all-trans-retinal. When used in parallel, these methods report both the actual amount of arrestin bound to the membrane surface and the functional aspects of arrestin binding, such as which arrestin loops are engaged and whether Meta II is stabilized. Most of these methods can also be applied to recombinant receptor reconstituted into liposomes, bicelles, and nanodisks.

LDRD final report on light-powered nanovehicles.

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

Shelnutt, John Allen; van Swol, Frank B.; Miller, James Edward

2003-11-01

We have investigated the possibility of constructing nanoscale metallic vehicles powered by biological motors or flagella that are activated and powered by visible light. The vehicle's body is to be composed of the surfactant bilayer of a liposome coated with metallic nanoparticles or nanosheets grown together into a porous single crystal. The diameter of the rigid metal vesicles is from about 50 nm to microns. Illumination with visible light activates a photosynthetic system in the bilayer that can generate a pH gradient across the liposomal membrane. The proton gradient can fuel a molecular motor that is incorporated into the membrane.more » Some molecular motors require ATP to fuel active transport. The protein ATP synthase, when embedded in the membrane, will use the pH gradient across the membrane to produce ATP from ADP and inorganic phosphate. The nanoscale vehicle is thus composed of both natural biological components (ATPase, flagellum; actin-myosin, kinesin-microtubules) and biomimetic components (metal vehicle casing, photosynthetic membrane) as functional units. Only light and storable ADP, phosphate, water, and weak electron donor are required fuel components. These nano-vehicles are being constructed by self-assembly and photocatalytic and autocatalytic reactions. The nano-vehicles can potentially respond to chemical gradients and other factors such as light intensity and field gradients, in a manner similar to the way that magnetic bacteria navigate. The delivery package might include decision-making and guidance components, drugs or other biological and chemical agents, explosives, catalytic reactors, and structural materials. We expected in one year to be able only to assess the problems and major issues at each stage of construction of the vehicle and the likely success of fabricating viable nanovehicles with our biomimetic photocatalytic approach. Surprisingly, we have been able to demonstrate that metallized photosynthetic liposomes can indeed be made. We have completed the synthesis of metallized liposomes with photosynthetic function included and studied these structures by electron microscopy. Both platinum and palladium nanosheeting have been used to coat the micelles. The stability of the vehicles to mechanical stress and the solution environment is enhanced by the single-crystalline platinum or palladium coating on the vesicle. With analogous platinized micelles, it is possible to dry the vehicles and re-suspend them with full functionality. However, with the liposomes drying on a TEM grid may cause the platinized liposomes to collapse, although probably stay viable in solution. It remains to be shown whether a proton motive force across the metallized bilayer membrane can be generated and whether we will also be able to incorporate various functional capabilities including ATP synthesis and functional molecular motors. Future tasks to complete the nanovehicles would be the incorporation of ATP synthase into metallized liposomes and the incorporation of a molecular motor into metallized liposomes.« less

Bile acids modulate signaling by functional perturbation of plasma membrane domains.

PubMed

Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

2013-12-13

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

New physiologically-relevant liver tissue model based on hierarchically cocultured primary rat hepatocytes with liver endothelial cells.

PubMed

Xiao, Wenjin; Perry, Guillaume; Komori, Kikuo; Sakai, Yasuyuki

2015-11-01

To develop an in vitro liver tissue equivalent, hepatocytes should be cocultured with liver non-parenchymal cells to mimic the in vivo physiological microenvironments. In this work, we describe a physiologically-relevant liver tissue model by hierarchically organizing layers of primary rat hepatocytes and human liver sinusoidal endothelial cells (TMNK-1) on an oxygen-permeable polydimethylsiloxane (PDMS) membrane, which facilitates direct oxygenation by diffusion through the membrane. This in vivo-mimicking hierarchical coculture was obtained by simply proceeding the overlay of TMNK-1 cells on the hepatocyte layer re-formed on the collagen immobilized PDMS membranes. The comparison of hepatic functionalities was achieved between coculture and sandwich culture with Matrigel, in the presence and absence of direct oxygenation. A complete double-layered structure of functional liver cells with vertical contact between hepatocytes and TMNK-1 was successfully constructed in the coculture with direct oxygen supply and was well-maintained for 14 days. The hepatocytes in this hierarchical culture exhibited improved survival, functional bile canaliculi formation, cellular level polarization and maintenance of metabolic activities including Cyp1A1/2 activity and albumin production. By contrast, the two cell populations formed discontinuous monolayers on the same surfaces in the non-oxygen-permeable cultures. These results demonstrate that (i) the direct oxygenation through the PDMS membranes enables very simple formation of a hierarchical structure consisting of a hepatocyte layer and a layer of TMNK-1 and (ii) we may include other non-parenchymal cells in this format easily, which can be widely applicable to other epithelial organs.

Artificial transmembrane ion channels from self-assembling peptide nanotubes

NASA Astrophysics Data System (ADS)

Ghadiri, M. Reza; Granja, Juan R.; Buehler, Lukas K.

1994-05-01

NATURALLY occurring membrane channels and pores are formed from a large family of diverse proteins, peptides and organic secon-dary metabolites whose vital biological functions include control of ion flow, signal transduction, molecular transport and produc-tion of cellular toxins. But despite the availability of a large amount of biochemical information about these molecules1, the design and synthesis of artificial systems that can mimic the bio-logical function of natural compounds remains a formidable task2-12. Here we present a simple strategy for the design of artifi-cial membrane ion channels based on a self-assembled cylindrical β-sheet peptide architecture13. Our systems-essentially stacks of peptide rings-display good channel-mediated ion-transport activ-ity with rates exceeding 107 ions s-1, rivalling the performance of many naturally occurring counterparts. Such molecular assemblies should find use in the design of novel cytotoxic agents, membrane transport vehicles and drug-delivery systems.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging.

PubMed

Zhang, Nan; Khan, Liakot A; Membreno, Edward; Jafari, Gholamali; Yan, Siyang; Zhang, Hongjie; Gobel, Verena

2017-10-03

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C. elegans intestine as a model for the analysis of polarized membrane biogenesis during tube morphogenesis, with emphasis on apical membrane and lumen biogenesis. The C. elegans twenty-cell single-layered intestinal epithelium is arranged into a simple bilaterally symmetrical tube, permitting analysis on a single-cell level. Membrane polarization occurs concomitantly with polarized cell division and migration during early embryogenesis, but de novo polarized membrane biogenesis continues throughout larval growth, when cells no longer proliferate and move. The latter setting allows one to separate subcellular changes that simultaneously mediate these different polarizing processes, difficult to distinguish in most polarity models. Apical-, basolateral membrane-, junctional-, cytoskeletal- and endomembrane components can be labeled and tracked throughout development by GFP fusion proteins, or assessed by in situ antibody staining. Together with the organism's genetic versatility, the C. elegans intestine thus provides a unique in vivo model for the visual, developmental, and molecular genetic analysis of polarized membrane and tube biogenesis. The specific methods (all standard) described here include how to: label intestinal subcellular components by antibody staining; analyze genes involved in polarized membrane biogenesis by loss-of-function studies adapted to the typically essential tubulogenesis genes; assess polarity defects during different developmental stages; interpret phenotypes by epifluorescence, differential interference contrast (DIC) and confocal microscopy; quantify visual defects. This protocol can be adapted to analyze any of the often highly conserved molecules involved in epithelial polarity, membrane biogenesis, tube and lumen morphogenesis.

Carbon Dioxide Separation Using Thermally Optimized Membranes

NASA Astrophysics Data System (ADS)

Young, J. S.; Jorgensen, B. S.; Espinoza, B. F.; Weimer, M. W.; Jarvinen, G. D.; Greenberg, A.; Khare, V.; Orme, C. J.; Wertsching, A. K.; Peterson, E. S.; Hopkins, S. D.; Acquaviva, J.

2002-05-01

The purpose of this project is to develop polymeric-metallic membranes for carbon dioxide separations that operate under a broad range of industrially relevant conditions not accessible with present membrane units. The last decade has witnessed a dramatic increase in the use of polymer membranes as an effective, economic and flexible tool for many commercial gas separations including air separation, the recovery of hydrogen from nitrogen, carbon monoxide, and methane mixtures, and the removal of carbon dioxide from natural gas. In each of these applications, high fluxes and excellent selectivities have relied on glassy polymer membranes which separate gases based on both size and solubility differences. To date, however, this technology has focused on optimizing materials for near ambient conditions. The development of polymeric materials that achieve the important combination of high selectivity, high permeability, and mechanical stability at temperatures significantly above 25oC and pressures above 10 bar, respectively, has been largely ignored. Consequently, there is a compelling rationale for the exploration of a new realm of polymer membrane separations. Indeed, the development of high temperature polymeric-metallic composite membranes for carbon dioxide separation at temperatures of 100-450 oC and pressures of 10-150 bar would provide a pivotal contribution with both economic and environmental benefits. Progress to date includes the first ever fabrication of a polymeric-metallic membrane that is selective from room temperature to 370oC. This achievement represents the highest demonstrated operating temperature at which a polymeric based membrane has successfully functioned. Additionally, we have generated the first polybenzamidizole silicate molecular composites. Finally, we have developed a technique that has enabled the first-ever simultaneous measurements of gas permeation and membrane compaction at elevated temperatures. This technique provides a unique approach to the optimization of long-term membrane performance under challenging operating conditions.

Permeability recovery of fouled forward osmosis membranes by chemical cleaning during a long-term operation of anaerobic osmotic membrane bioreactors treating low-strength wastewater.

PubMed

Wang, Xinhua; Hu, Taozhan; Wang, Zhiwei; Li, Xiufen; Ren, Yueping

2017-10-15

Anaerobic osmotic membrane bioreactor (AnOMBR) has gained increasing interests in wastewater treatment owing to its simultaneous recovery of biogas and water. However, the forward osmosis (FO) membrane fouling was severe during a long-term operation of AnOMBRs. Here, we aim to recover the permeability of fouled FO membranes by chemical cleaning. Specifically speaking, an optimal chemical cleaning procedure was searched for fouled thin film composite polyamide FO (TFC-FO) membranes in a novel microfiltration (MF) assisted AnOMBR (AnMF-OMBR). The results indicated that citric acid, disodium ethylenediaminetetraacetate (EDTA-2Na), hydrochloric acid (HCl), sodium dodecyl sulfate (SDS) and sodium hydroxide (NaOH) had a low cleaning efficiency of less than 15%, while hydrogen peroxide (H 2 O 2 ) could effectively remove foulants from the TFC-FO membrane surface (almost 100%) through oxidizing the functional group of the organic foulants and disintegrating the colloids and microbe flocs into fine particles. Nevertheless, the damage of H 2 O 2 to the TFC-FO membrane was observed when a high cleaning concentration and a long duration were applied. In this case, the optimal cleaning conditions including cleaning concentration and time for fouled TFC-FO membranes were selected through confocal laser scanning microscope (CLSM) and scanning electron microscopy (SEM) images and the flux recovery rate. The results suggested that the optimal cleaning procedure for fouled TFC-FO membranes was use of 0.5% H 2 O 2 at 25 °C for 6 h, and after that, the cleaned TFC-FO membrane had the same performance as a virgin one including water flux and rejection for organic matters and phosphorus during the operation of AnMF-OMBR. Copyright © 2017 Elsevier Ltd. All rights reserved.

Position-Dependent Diffusion Tensors in Anisotropic Media from Simulation: Oxygen Transport in and through Membranes.

PubMed

Ghysels, An; Venable, Richard M; Pastor, Richard W; Hummer, Gerhard

2017-06-13

A Bayesian-based methodology is developed to estimate diffusion tensors from molecular dynamics simulations of permeants in anisotropic media, and is applied to oxygen in lipid bilayers. By a separation of variables in the Smoluchowski diffusion equation, the multidimensional diffusion is reduced to coupled one-dimensional diffusion problems that are treated by discretization. The resulting diffusivity profiles characterize the membrane transport dynamics as a function of the position across the membrane, discriminating between diffusion normal and parallel to the membrane. The methodology is first validated with neat water, neat hexadecane, and a hexadecane slab surrounded by water, the latter being a simple model for a lipid membrane. Next, a bilayer consisting of pure 1-palmitoyl 2-oleoylphosphatidylcholine (POPC), and a bilayer mimicking the lipid composition of the inner mitochondrial membrane, including cardiolipin, are investigated. We analyze the detailed time evolution of oxygen molecules, in terms of both normal diffusion through and radial diffusion inside the membrane. Diffusion is fast in the more loosely packed interleaflet region, and anisotropic, with oxygen spreading more rapidly in the membrane plane than normal to it. Visualization of the propagator shows that oxygen enters the membrane rapidly, reaching its thermodynamically favored center in about 1 ns, despite the free energy barrier at the headgroup region. Oxygen transport is quantified by computing the oxygen permeability of the membranes and the average radial diffusivity, which confirm the anisotropy of the diffusion. The position-dependent diffusion constants and free energies are used to construct compartmental models and test assumptions used in estimating permeability, including Overton's rule. In particular, a hexadecane slab surrounded by water is found to be a poor model of oxygen transport in membranes because the relevant energy barriers differ substantially.

Exploring Molecular-Biomembrane Interactions with Surface Plasmon Resonance and Dual Polarization Interferometry Technology: Expanding the Spotlight onto Biomembrane Structure.

PubMed

Lee, Tzong-Hsien; Hirst, Daniel J; Kulkarni, Ketav; Del Borgo, Mark P; Aguilar, Marie-Isabel

2018-06-13

The molecular analysis of biomolecular-membrane interactions is central to understanding most cellular systems but has emerged as a complex technical challenge given the complexities of membrane structure and composition across all living cells. We present a review of the application of surface plasmon resonance and dual polarization interferometry-based biosensors to the study of biomembrane-based systems using both planar mono- or bilayers or liposomes. We first describe the optical principals and instrumentation of surface plasmon resonance, including both linear and extraordinary transmission modes and dual polarization interferometry. We then describe the wide range of model membrane systems that have been developed for deposition on the chips surfaces that include planar, polymer cushioned, tethered bilayers, and liposomes. This is followed by a description of the different chemical immobilization or physisorption techniques. The application of this broad range of engineered membrane surfaces to biomolecular-membrane interactions is then overviewed and how the information obtained using these techniques enhance our molecular understanding of membrane-mediated peptide and protein function. We first discuss experiments where SPR alone has been used to characterize membrane binding and describe how these studies yielded novel insight into the molecular events associated with membrane interactions and how they provided a significant impetus to more recent studies that focus on coincident membrane structure changes during binding of peptides and proteins. We then discuss the emerging limitations of not monitoring the effects on membrane structure and how SPR data can be combined with DPI to provide significant new information on how a membrane responds to the binding of peptides and proteins.

Biological effects of electric shock and heat denaturation and oxidation of molecules, membranes, and cellular functions.

PubMed

Tsong, T Y; Su, Z D

1999-10-30

Direct exposure of cells in suspension to intense electric pulses is known to produce damages to cell membranes and supramolecular organizations of cells, and denaturation of macromolecules, much like injuries and tears seen in electric trauma patients. Thus, the system has been used as a laboratory model for investigating the biochemical basis of electric injury. An intense electric pulse can produce two major effects on cells--one caused by the field, or the electric potential, and the other by current, or the electric energy. The field-induced transmembrane potential can produce electro-conformational changes of ion channels and ion pumps and, when the potential exceeds the dielectric strength of the cell membrane (approximately 500 mV for a pulse width of a few ms), electro-conformational damages and electroporations of membrane proteins and lipid bilayers. These events lead to passage of electric current through the membrane-porated cells and to heating of cell membranes and cytoplasmic contents. The subsequent denaturation of cell membranes and cytoplasmic macromolecules brings about many complex biochemical reactions, including oxidation of proteins and lipids. The combined effects may cripple the cells beyond repair. This communication will focus on the thermal effects of electric shock. After a brief review of the current state of knowledge on thermal denaturation of soluble enzymes and muscle proteins, this paper will describe experiments on the thermal denaturation of cellular components and functions, such as nucleosomes, and the electron transport chain and ATP synthetic enzymes of the mitochondrial inner membranes. Data will show that lipid peroxidation and the subsequent loss of the energy-transducing ability of the cells may occur even at moderate temperatures between 40 degrees C and 45 degrees C. However, lipid peroxidation may be prevented with reducing reagents such as mercaptoethanol, dithiothreitol, and ascorbic acid. Reactivation of denatured cellular proteins and functions may also be possible and a strategy for doing so is discussed.

Molecular transport properties through carbon nanotube membrane

NASA Astrophysics Data System (ADS)

Majumder, Mainak

Molecular transport through hollow cores of crystalline carbon nanotubes (CNTs) are of considerable interest from the fundamental and application point of view. This dissertation focuses on understanding molecular transport through a membrane platform consisting of open ended CNTs with ˜ 7 nm core diameter and ˜ 1010 CNTs/cm2 encapsulated in an inert polymer matrix. While ionic diffusion through the membrane is close to bulk diffusion expectations, gases and liquids were respectively observed to be transported ˜ 10 times faster than Knudsen diffusion and ˜ 10000--100000 times faster than hydrodynamic flow predictions. This phenomenon has been attributed to the non-interactive and frictionless graphitic interface. Functionalization of the CNT tips was observed to change selectivity and flux through the CNT membranes with analogy to 'gate-keeper' functionality in biological membranes. An electro-chemical diazonium grafting chemistry was utilized for enhancing the functional density on the CNT membranes. A strategy to confine the reactions at the CNT tips by a fast flowing liquid column was also designed. Characterization using electrochemical impedance spectroscopy and dye assay indicated ˜ 5--6 times increase in functional density. Electrochemical impedance spectroscopy experiments on CNT membrane/electrode functionalized with charged macro-molecules showed voltage-controlled conformational change. Similar chemistry has been applied for realizing 'voltage-gated' transport channels with potential application in trans-dermal drug delivery. Electrically-facilitated transport (a geometry in which an electric field gradient acts across the membrane) through the CNT and functionalized CNT membranes was observed to be electrosmotically controlled. Finally, a simulation framework based on continuum electrostatics and finite elements has been developed to further the understanding of transport through the CNT membranes. KEYWORDS: carbon nanotube membrane, nano-scale hydrodynamics, diazonium grafting, voltage-gated membrane, finite element analysis

High performance, durable polymers including poly(phenylene)

DOEpatents

Fujimoto, Cy; Pratt, Harry; Anderson, Travis Mark

2017-02-28

The present invention relates to functionalized polymers including a poly(phenylene) structure. In some embodiments, the polymers and copolymers of the invention include a highly localized concentration of acidic moieties, which facilitate proton transport and conduction through networks formed from these polymers. In addition, the polymers can include functional moieties, such as electron-withdrawing moieties, to protect the polymeric backbone, thereby extending its durability. Such enhanced proton transport and durability can be beneficial for any high performance platform that employs proton exchange polymeric membranes, such as in fuel cells or flow batteries.

Structurally distinct membrane nanotubes between human macrophages support long-distance vesicular traffic or surfing of bacteria.

PubMed

Onfelt, Björn; Nedvetzki, Shlomo; Benninger, Richard K P; Purbhoo, Marco A; Sowinski, Stefanie; Hume, Alistair N; Seabra, Miguel C; Neil, Mark A A; French, Paul M W; Davis, Daniel M

2006-12-15

We report that two classes of membrane nanotubes between human monocyte-derived macrophages can be distinguished by their cytoskeletal structure and their functional properties. Thin membrane nanotubes contained only F-actin, whereas thicker nanotubes, i.e., those > approximately 0.7 microm in diameter, contained both F-actin and microtubules. Bacteria could be trapped and surf along thin, but not thick, membrane nanotubes toward connected macrophage cell bodies. Once at the cell body, bacteria could then be phagocytosed. The movement of bacteria is aided by a constitutive flow of the nanotube surface because streptavidin-coated beads were similarly able to traffic along nanotubes between surface-biotinylated macrophages. Mitochondria and intracellular vesicles, including late endosomes and lysosomes, could be detected within thick, but not thin, membrane nanotubes. Analysis from kymographs demonstrated that vesicles moved in a stepwise, bidirectional manner at approximately 1 microm/s, consistent with their traffic being mediated by the microtubules found only in thick nanotubes. Vesicular traffic in thick nanotubes and surfing of beads along thin nanotubes were both stopped upon the addition of azide, demonstrating that both processes require ATP. However, microtubule destabilizing agents colchicine or nocodazole abrogated vesicular transport but not the flow of the nanotube surface, confirming that distinct cytoskeletal structures of nanotubes give rise to different functional properties. Thus, membrane nanotubes between macrophages are more complex than unvarying ubiquitous membrane tethers and facilitate several means for distal interactions between immune cells.

Design and function of biomimetic multilayer water purification membranes

PubMed Central

Ling, Shengjie; Qin, Zhao; Huang, Wenwen; Cao, Sufeng; Kaplan, David L.; Buehler, Markus J.

2017-01-01

Multilayer architectures in water purification membranes enable increased water throughput, high filter efficiency, and high molecular loading capacity. However, the preparation of membranes with well-organized multilayer structures, starting from the nanoscale to maximize filtration efficiency, remains a challenge. We report a complete strategy to fully realize a novel biomaterial-based multilayer nanoporous membrane via the integration of computational simulation and experimental fabrication. Our comparative computational simulations, based on coarse-grained models of protein nanofibrils and mineral plates, reveal that the multilayer structure can only form with weak interactions between nanofibrils and mineral plates. We demonstrate experimentally that silk nanofibril (SNF) and hydroxyapatite (HAP) can be used to fabricate highly ordered multilayer membranes with nanoporous features by combining protein self-assembly and in situ biomineralization. The production is optimized to be a simple and highly repeatable process that does not require sophisticated equipment and is suitable for scaled production of low-cost water purification membranes. These membranes not only show ultrafast water penetration but also exhibit broad utility and high efficiency of removal and even reuse (in some cases) of contaminants, including heavy metal ions, dyes, proteins, and other nanoparticles in water. Our biomimetic design and synthesis of these functional SNF/HAP materials have established a paradigm that could lead to the large-scale, low-cost production of multilayer materials with broad spectrum and efficiency for water purification, with applications in wastewater treatment, biomedicine, food industry, and the life sciences. PMID:28435877

Surface-functionalized nanoparticle permeation triggers lipid displacement and water and ion leakage.

PubMed

Oroskar, Priyanka A; Jameson, Cynthia J; Murad, Sohail

2015-01-27

Functionalized nanoparticles (NPs) are considered suitable carriers for targeted drug delivery systems. However, the ion and water leakage induced by permeation of these nanoparticles is a challenge in these drug delivery methods because of cytotoxic effects of some ions. In this study, we have carried out a series of coarse-grained molecular dynamics simulations to investigate the effect of length of ligands on permeation of a nanoparticle across a protein-free phospholipid bilayer membrane. Water and ion penetration as well as incidence of lipid flip-flop events and loss of lipid molecules from the membrane are explored in this study while varying the nanoparticle size, length of ligand, ion concentration gradient, pressure differential across the membrane, and nanoparticle permeation velocity. Some results from our studies include (1) the number of water molecules in the interior of the membrane during ligand-coated nanoparticle permeation increases with nanoparticle size, ligand length, pressure differential, and permeation velocity but is not sensitive to the ion concentration gradient; (2) some lipid molecules leave the membrane by being entangled with ligands of the NP instead of completing the flip-flop that permits them to rejoin the membrane, thereby leading to fewer flip-flop events; and (3) the formation of water columns or water "fingers" provides a mechanism of ion transport across lipid bilayer membranes, but such ion penetration events are less likely for sodium ions than chloride ions and less likely for nanoparticles with longer-ligands.

Design and function of biomimetic multilayer water purification membranes.

PubMed

Ling, Shengjie; Qin, Zhao; Huang, Wenwen; Cao, Sufeng; Kaplan, David L; Buehler, Markus J

2017-04-01

Multilayer architectures in water purification membranes enable increased water throughput, high filter efficiency, and high molecular loading capacity. However, the preparation of membranes with well-organized multilayer structures, starting from the nanoscale to maximize filtration efficiency, remains a challenge. We report a complete strategy to fully realize a novel biomaterial-based multilayer nanoporous membrane via the integration of computational simulation and experimental fabrication. Our comparative computational simulations, based on coarse-grained models of protein nanofibrils and mineral plates, reveal that the multilayer structure can only form with weak interactions between nanofibrils and mineral plates. We demonstrate experimentally that silk nanofibril (SNF) and hydroxyapatite (HAP) can be used to fabricate highly ordered multilayer membranes with nanoporous features by combining protein self-assembly and in situ biomineralization. The production is optimized to be a simple and highly repeatable process that does not require sophisticated equipment and is suitable for scaled production of low-cost water purification membranes. These membranes not only show ultrafast water penetration but also exhibit broad utility and high efficiency of removal and even reuse (in some cases) of contaminants, including heavy metal ions, dyes, proteins, and other nanoparticles in water. Our biomimetic design and synthesis of these functional SNF/HAP materials have established a paradigm that could lead to the large-scale, low-cost production of multilayer materials with broad spectrum and efficiency for water purification, with applications in wastewater treatment, biomedicine, food industry, and the life sciences.

Dynamic protein S-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence.

PubMed

Brown, Robert W B; Sharma, Aabha I; Engman, David M

2017-04-01

Eukaryotic parasites possess complex life cycles and utilize an assortment of molecular mechanisms to overcome physical barriers, suppress and/or bypass the host immune response, including invading host cells where they can replicate in a protected intracellular niche. Protein S-palmitoylation is a dynamic post-translational modification in which the fatty acid palmitate is covalently linked to cysteine residues on proteins by the enzyme palmitoyl acyltransferase (PAT) and can be removed by lysosomal palmitoyl-protein thioesterase (PPT) or cytosolic acyl-protein thioesterase (APT). In addition to anchoring proteins to intracellular membranes, functions of dynamic palmitoylation include - targeting proteins to specific intracellular compartments via trafficking pathways, regulating the cycling of proteins between membranes, modulating protein function and regulating protein stability. Recent studies in the eukaryotic parasites - Plasmodium falciparum, Toxoplasma gondii, Trypanosoma brucei, Cryptococcus neoformans and Giardia lamblia - have identified large families of PATs and palmitoylated proteins. Many palmitoylated proteins are important for diverse aspects of pathogenesis, including differentiation into infective life cycle stages, biogenesis and tethering of secretory organelles, assembling the machinery powering motility and targeting virulence factors to the plasma membrane. This review aims to summarize our current knowledge of palmitoylation in eukaryotic parasites, highlighting five exemplary mechanisms of parasite virulence dependent on palmitoylation.

Membrane tension and cytoskeleton organization in cell motility.

PubMed

Sens, Pierre; Plastino, Julie

2015-07-15

Cell membrane shape changes are important for many aspects of normal biological function, such as tissue development, wound healing and cell division and motility. Various disease states are associated with deregulation of how cells move and change shape, including notably tumor initiation and cancer cell metastasis. Cell motility is powered, in large part, by the controlled assembly and disassembly of the actin cytoskeleton. Much of this dynamic happens in close proximity to the plasma membrane due to the fact that actin assembly factors are membrane-bound, and thus actin filaments are generally oriented such that their growth occurs against or near the membrane. For a long time, the membrane was viewed as a relatively passive scaffold for signaling. However, results from the last five years show that this is not the whole picture, and that the dynamics of the actin cytoskeleton are intimately linked to the mechanics of the cell membrane. In this review, we summarize recent findings concerning the role of plasma membrane mechanics in cell cytoskeleton dynamics and architecture, showing that the cell membrane is not just an envelope or a barrier for actin assembly, but is a master regulator controlling cytoskeleton dynamics and cell polarity.

Systems biology of cellular membranes: a convergence with biophysics.

PubMed

Chabanon, Morgan; Stachowiak, Jeanne C; Rangamani, Padmini

2017-09-01

Systems biology and systems medicine have played an important role in the last two decades in shaping our understanding of biological processes. While systems biology is synonymous with network maps and '-omics' approaches, it is not often associated with mechanical processes. Here, we make the case for considering the mechanical and geometrical aspects of biological membranes as a key step in pushing the frontiers of systems biology of cellular membranes forward. We begin by introducing the basic components of cellular membranes, and highlight their dynamical aspects. We then survey the functions of the plasma membrane and the endomembrane system in signaling, and discuss the role and origin of membrane curvature in these diverse cellular processes. We further give an overview of the experimental and modeling approaches to study membrane phenomena. We close with a perspective on the converging futures of systems biology and membrane biophysics, invoking the need to include physical variables such as location and geometry in the study of cellular membranes. WIREs Syst Biol Med 2017, 9:e1386. doi: 10.1002/wsbm.1386 For further resources related to this article, please visit the WIREs website. © 2017 Wiley Periodicals, Inc.

The Urethral Rhabdosphincter, Levator Ani Muscle, and Perineal Membrane: A Review

PubMed Central

Hinata, Nobuyuki; Murakami, Gen

2014-01-01

Detailed knowledge of the anatomy of the rhabdosphincter and adjacent tissues is mandatory during urologic surgery to ensure reliable oncologic and functional outcomes. To characterize the levator ani (LA) function for the urethral sphincter, we described connective tissue morphology between the LA and urethral rhabdosphincter. The interface tissue between the LA and rhabdosphincter area in males contained abundant irregularly arrayed elastic fibers and smooth muscles. The male rhabdosphincter was positioned alongside the LA to divide the elevation force and not in-series along the axis of LA contraction. The male perineal membrane was thin but solid and extends along the inferior margin or bottom of the rhabdosphincter area. In contrast, the female rhabdosphincter, including the compressor urethrae and urethrovaginal sphincter muscles, was embedded in the elastic fiber mesh that is continuous with the thick, multilaminar perineal membrane. The inferomedial edge of the female LA was attached to the upper surface of the perineal membrane and not directly attached to the rhabdosphincter. We presented new diagrams showing the gender differences in topographical anatomy of the LA and rhabdosphincter. PMID:24877147

The membrane fusion enigma: SNAREs, Sec1/Munc18 proteins, and their accomplices--guilty as charged?

PubMed

Rizo, Josep; Südhof, Thomas C

2012-01-01

Neurotransmitter release is governed by proteins that have homo-logs in most types of intracellular membrane fusion, including the Sec1/Munc18 protein Munc18-1 and the SNARE proteins syntaxin-1, synaptobrevin/VAMP, and SNAP-25. The SNAREs initiate fusion by forming tight SNARE complexes that bring the vesicle and plasma membranes together. SNARE maintenance in a functional state depends on two chaperone systems (Hsc70/αCSP/SGT and synuclein); defects in these systems lead to neurodegeneration. Munc18-1 binds to an autoinhibitory closed conformation of syntaxin-1, gating formation of SNARE complexes, and also binds to SNARE complexes, which likely underlies the crucial function of Munc18-1 in membrane fusion by an as-yet unclear mechanism. Syntaxin-1 opening is mediated by Munc13s through their MUN domain, which is homologous to diverse tethering factors and may also have a general role in fusion. MUN domain activity is likely modulated in diverse presynaptic plasticity processes that depend on Ca(2+) and RIM proteins, among others.

The First Cell Membranes

NASA Technical Reports Server (NTRS)

Deamer, David; Dworkin, Jason P.; Sandford, Scott A.; Bernstein, Max P.; Allamandola, Louis J.

2004-01-01

Organic compounds are synthesized in the interstellar medium and can be delivered to planetary surfaces such as the early Earth, where they mix with endogenous organic mixtures. Some of these compounds are amphiphilic, having polar and non-polar groups on the same molecule. Amphiphilic compounds spontaneously self-assembly into more complex structures such as bimolecular layers, which in turn form closed membranous vesicles. The first forms of cellular life required self-assembled membranes that were likely to be available on the prebiotic Earth. Laboratory simulations show that such vesicles readily encapsulate functional macromolecules, including nucleic acids and polymerases. A goal of future investigations is to fabricate artificial cells as models of the origin of life.

Membrane microparticles and diseases.

PubMed

Wu, Z-H; Ji, C-L; Li, H; Qiu, G-X; Gao, C-J; Weng, X-S

2013-09-01

Membrane microparticles (MPs) are plasma membrane-derived vesicles shed by various types of activated or apoptotic cells including platelets, monocytes, endothelial cells, red blood cells, and granulocytes. MPs are being increasingly recognized as important regulators of cell-to-cell interactions. Recent evidences suggest they may play important functions not only in homeostasis but also in the pathogenesis of a number of diseases such as vascular diseases, cancer, infectious diseases and diabetes mellitus. Accordingly, inhibiting the production of MPs may serve as a novel therapeutic strategy for these diseases. Here we review recent advances on the mechanism underlying the generation of MPs and the role of MPs in vascular diseases, cancer, diabetes, inflammation, and pathogen infection.

Hierarchical multiscale hyperporous block copolymer membranes via tunable dual-phase separation

PubMed Central

Yoo, Seungmin; Kim, Jung-Hwan; Shin, Myoungsoo; Park, Hyungmin; Kim, Jeong-Hoon; Lee, Sang-Young; Park, Soojin

2015-01-01

The rational design and realization of revolutionary porous structures have been long-standing challenges in membrane science. We demonstrate a new class of amphiphilic polystyrene-block-poly(4-vinylpyridine) block copolymer (BCP)–based porous membranes featuring hierarchical multiscale hyperporous structures. The introduction of surface energy–modifying agents and the control of major phase separation parameters (such as nonsolvent polarity and solvent drying time) enable tunable dual-phase separation of BCPs, eventually leading to macro/nanoscale porous structures and chemical functionalities far beyond those accessible with conventional approaches. Application of this BCP membrane to a lithium-ion battery separator affords exceptional improvement in electrochemical performance. The dual-phase separation–driven macro/nanopore construction strategy, owing to its simplicity and tunability, is expected to be readily applicable to a rich variety of membrane fields including molecular separation, water purification, and energy-related devices. PMID:26601212

Prominin-1-containing membrane vesicles: origins, formation, and utility.

PubMed

Marzesco, Anne-Marie

2013-01-01

The stem cell antigen prominin-1 (CD133) is associated with two major types (small and large) of extracellular membrane vesicles in addition to its selective concentration in various kinds of plasma membrane protrusion. During development of the mammalian central nervous system, differentiating neuroepithelial stem cells release these vesicles into the embryonic cerebrospinal fluid. In glioblastoma patients, an increase of such vesicles, particularly the smaller ones, have been also observed in cerebrospinal fluid. Similarly, hematopoietic stem and progenitor cells release small ones concomitantly with their differentiation. Although the functional significance of these prominin-1-containing membrane vesicles is poorly understood, a link between differentiation of stem (and cancer stem) cells and their release is emerging. In this chapter, I will summarize our knowledge about prominin-1-containing membrane vesicles including a potential role in cell-cell communication and highlight their prospective value as a new biomarker for tumorigenesis diagnostics.

Ubiquitin-dependent sorting of integral membrane proteins for degradation in lysosomes

PubMed Central

Piper, Robert C.

2007-01-01

Summary The pathways that deliver newly synthesized proteins that reside in lysosomes are well understood by comparison with our knowledge of how integral membrane proteins are sorted and delivered to the lysosome for degradation. Many membrane proteins are sorted to lysosomes following ubiquitination, which provides a sorting signal that can operate for sorting at the TGN (trans-Golgi network), at the plasma membrane or at the endosome for delivery into lumenal vesicles. Candidate multicomponent machines that can potentially move ubiquitinated integral membrane cargo proteins have been identified, but much work is still required to ascertain which of these candidates directly recognizes ubiquitinated cargo and what they do with cargo after recognition. In the case of the machinery required for sorting into the lumenal vesicles of endosomes, other functions have also been determined including a link between sorting and movement of endosomes along microtubules. PMID:17689064

Ultrathin Ceramic Membranes as Scaffolds for Functional Cell Coculture Models on a Biomimetic Scale

PubMed Central

Jud, Corinne; Ahmed, Sher; Müller, Loretta; Kinnear, Calum; Vanhecke, Dimitri; Umehara, Yuki; Frey, Sabine; Liley, Martha; Angeloni, Silvia; Petri-Fink, Alke; Rothen-Rutishauser, Barbara

2015-01-01

Abstract Epithelial tissue serves as an interface between biological compartments. Many in vitro epithelial cell models have been developed as an alternative to animal experiments to answer a range of research questions. These in vitro models are grown on permeable two-chamber systems; however, commercially available, polymer-based cell culture inserts are around 10 μm thick. Since the basement membrane found in biological systems is usually less than 1 μm thick, the 10-fold thickness of cell culture inserts is a major limitation in the establishment of realistic models. In this work, an alternative insert, accommodating an ultrathin ceramic membrane with a thickness of only 500 nm (i.e., the Silicon nitride Microporous Permeable Insert [SIMPLI]-well), was produced and used to refine an established human alveolar barrier coculture model by both replacing the conventional inserts with the SIMPLI-well and completing it with endothelial cells. The structural–functional relationship of the model was evaluated, including the translocation of gold nanoparticles across the barrier, revealing a higher translocation if compared to corresponding polyethylene terephthalate (PET) membranes. This study demonstrates the power of the SIMPLI-well system as a scaffold for epithelial tissue cell models on a truly biomimetic scale, allowing construction of more functionally accurate models of human biological barriers. PMID:26713225

Increased curvature of hollow fiber membranes could up-regulate differential functions of renal tubular cell layers.

PubMed

Shen, Chong; Meng, Qin; Zhang, Guoliang

2013-08-01

Tissue engineering devices as in vitro cell culture systems in scaffolds has encountered the bottleneck due to their much lower cell functions than real tissues/organs in vivo. Such situation has been improved in some extent by mimicking the cell microenvironments in vivo from either chemical or physical ways. However, microenvironmental curvature, commonly seen in real tissues/organs, has never been manipulated to regulate the cell performance in vitro. In this regard, this paper fabricated polysulfone membranes with or without polyethylene glycol modification to investigate the impact of curvature on two renal tubular cells. Regardless the varying membrane curvatures among hollow fiber membranes of different diameters and flat membrane of zero curvature, both renal cells could well attach at 4 h of seeding and form similar confluent layers at 6 days on each membrane. Nevertheless, the renal cells on hollow fibers, though showing confluent morphology as those on flat membranes, expressed higher renal functions and, moreover, the renal functions significantly increased with the membrane curvature among hollow fibers. Such upregulation on functions was unassociated with mass transport barrier of hollow fibers, because the cultures on lengthwise cut hollow fibers without mass transfer barrier showed same curvature effect on renal functions as whole hollow fibers. It could be proposed that the curvature of hollow fiber membrane approaching to the large curvature in kidney tubules increased the mechanical stress in the renal cells and thus might up-regulate the renal cell functions. In conclusion, the increase of substrate curvature could up-regulate the cell functions without altering the confluent cell morphology and this finding will facilitate the design of functional tissue engineering devices. Copyright © 2013 Wiley Periodicals, Inc.

The scope of phage display for membrane proteins.

PubMed

Vithayathil, Rosemarie; Hooy, Richard M; Cocco, Melanie J; Weiss, Gregory A

2011-12-09

Numerous examples of phage display applied to soluble proteins demonstrate the power of the technique for protein engineering, affinity reagent discovery and structure-function studies. Recent reports have expanded phage display to include membrane proteins (MPs). The scope and limitations of MP display remain undefined. Therefore, we report data from the phage display of representative types of membrane-associated proteins including plasma, nuclear, peripheral, single and multipass. The peripheral MP neuromodulin displays robustly with packaging by conventional M13-KO7 helper phage. The monotopic MP Nogo-66 can also display on the phage surface, if packaged by the modified M13-KO7(+) helper phage. The modified phage coat of KO7(+) can better mimic the zwitterionic character of the plasma membrane. Four examples of putatively α-helical, integral MPs failed to express as fusions to an anchoring phage coat protein and therefore did not display on the phage surface. However, the β-barrel MPs ShuA (Shigella heme uptake A) and MOMP (major outer membrane protein), which pass through the membrane 22 and 16 times, respectively, can display surprisingly well on the surfaces of both conventional and KO7(+) phages. The results provide a guide for protein engineering and large-scale mutagenesis enabled by the phage display of MPs. Copyright © 2011 Elsevier Ltd. All rights reserved.

Proteome-wide identification of predominant subcellular protein localizations in a bacterial model organism

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

Stekhoven, Daniel J.; Omasits, Ulrich; Quebatte, Maxime

2014-03-01

Proteomics data provide unique insights into biological systems, including the predominant subcellular localization (SCL) of proteins, which can reveal important clues about their functions. Here we analyzed data of a complete prokaryotic proteome expressed under two conditions mimicking interaction of the emerging pathogen Bartonella henselae with its mammalian host. Normalized spectral count data from cytoplasmic, total membrane, inner and outer membrane fractions allowed us to identify the predominant SCL for 82% of the identified proteins. The spectral count proportion of total membrane versus cytoplasmic fractions indicated the propensity of cytoplasmic proteins to co-fractionate with the inner membrane, and enabled usmore » to distinguish cytoplasmic, peripheral innermembrane and bona fide inner membrane proteins. Principal component analysis and k-nearest neighbor classification training on selected marker proteins or predominantly localized proteins, allowed us to determine an extensive catalog of at least 74 expressed outer membrane proteins, and to extend the SCL assignment to 94% of the identified proteins, including 18% where in silico methods gave no prediction. Suitable experimental proteomics data combined with straightforward computational approaches can thus identify the predominant SCL on a proteome-wide scale. Finally, we present a conceptual approach to identify proteins potentially changing their SCL in a condition-dependent fashion.« less

Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress.

PubMed

Pascua-Maestro, Raquel; Diez-Hermano, Sergio; Lillo, Concepción; Ganfornina, Maria D; Sanchez, Diego

2017-02-01

Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases. These results open therapeutic opportunities by providing a route of entry and a repair mechanism for lysosomes in pathological situations.

Membrane protein synthesis in cell-free systems: from bio-mimetic systems to bio-membranes.

PubMed

Sachse, Rita; Dondapati, Srujan K; Fenz, Susanne F; Schmidt, Thomas; Kubick, Stefan

2014-08-25

When taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Unique battery with an active membrane separator having uniform physico-chemically functionalized ion channels and a method making the same

DOEpatents

Gerald, II, Rex E.; Ruscic, Katarina J [Chicago, IL; Sears, Devin N [Spruce Grove, CA; Smith, Luis J [Natick, MA; Klingler, Robert J [Glenview, IL; Rathke, Jerome W [Homer Glen, IL

2012-02-21

The invention relates to a unique battery having an active, porous membrane and method of making the same. More specifically the invention relates to a sealed battery system having a porous, metal oxide membrane with uniform, physicochemically functionalized ion channels capable of adjustable ionic interaction. The physicochemically-active porous membrane purports dual functions: an electronic insulator (separator) and a unidirectional ion-transporter (electrolyte). The electrochemical cell membrane is activated for the transport of ions by contiguous ion coordination sites on the interior two-dimensional surfaces of the trans-membrane unidirectional pores. The membrane material is designed to have physicochemical interaction with ions. Control of the extent of the interactions between the ions and the interior pore walls of the membrane and other materials, chemicals, or structures contained within the pores provides adjustability of the ionic conductivity of the membrane.

Effect of garlic-derived organosulfur compounds on mitochondrial function and integrity in isolated mouse liver mitochondria.

PubMed

Caro, Andres A; Adlong, Luke W; Crocker, Samuel J; Gardner, Michael W; Luikart, Emily F; Gron, Liz U

2012-10-17

The objectives of this work were to evaluate the direct effects of diallysulfide (DAS) and diallyldisulfide (DADS), two major organosulfur compounds of garlic oil, on mitochondrial function and integrity, by using isolated mouse liver mitochondria in a cell-free system. DADS produced concentration-dependent mitochondrial swelling over the range 125-1000μM, while DAS was ineffective. Swelling experiments performed with de-energized or energized mitochondria showed similar maximal swelling amplitudes. Cyclosporin A (1μM), or ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, 1mM) were ineffective in inhibiting DADS-induced mitochondrial swelling. DADS produced a minor (12%) decrease in mitochondrial membrane protein thiols, but did not induce clustering of mitochondrial membrane proteins. Incubation of mitochondria with DADS (but not DAS) produced an increase in the oxidation rate of 2',7' dichlorofluorescein diacetate (DCFH-DA), together with depletion of reduced glutathione (GSH) and increased lipid peroxidation. DADS (but not DAS) produced a concentration-dependent dissipation of the mitochondrial membrane potential, but did not induce cytochrome c release. DADS-dependent effects, including mitochondrial swelling, DCFH-DA oxidation, lipid peroxidation and loss of mitochondrial membrane potential, were inhibited by antioxidants and iron chelators. These results suggest that DADS causes direct impairment of mitochondrial function as the result of oxidation of the membrane lipid phase initiated by the GSH- and iron-dependent generation of oxidants. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Challenges in the Development of Functional Assays of Membrane Proteins

PubMed Central

Tiefenauer, Louis; Demarche, Sophie

2012-01-01

Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

Possible role for fundus autofluorescence as a predictive factor for visual acuity recovery after epiretinal membrane surgery.

PubMed

Brito, Pedro N; Gomes, Nuno L; Vieira, Marco P; Faria, Pedro A; Fernandes, Augusto V; Rocha-Sousa, Amândio; Falcão-Reis, Fernando

2014-02-01

To study the potential association between fundus autofluorescence, spectral-domain optical coherence tomography, and visual acuity in patients undergoing surgery because of epiretinal membranes. Prospective, interventional case series including 26 patients submitted to vitrectomy because of symptomatic epiretinal membranes. Preoperative evaluation consisted of a complete ophthalmologic examination, autofluorescence, and spectral-domain optical coherence tomography. Studied variables included foveal autofluorescence (fov.AF), photoreceptor inner segment/outer segment (IS/OS) junction line integrity, external limiting membrane integrity, central foveal thickness, and foveal morphology. All examinations were repeated at the first, third, and sixth postoperative months. The main outcome measures were logarithm of minimal angle resolution visual acuity, fov.AF integrity, and IS/OS integrity. All cases showing a continuous IS/OS line had an intact fov.AF, whereas patients with IS/OS disruption could have either an increased area of foveal hypoautofluorescence or an intact fov.AF, with the latter being associated with IS/OS integrity recovery in follow-up spectral-domain optical coherence tomography imaging. The only preoperative variables presenting a significant correlation with final visual acuity were baseline visual acuity (P = 0.047) and fov.AF grade (P = 0.023). Recovery of IS/OS line integrity after surgery, in patients with preoperative IS/OS disruption and normal fov.AF, can be explained by the presence of a functional retinal pigment epithelium-photoreceptor complex, supporting normal photoreceptor activity. Autofluorescence imaging provides a functional component to the study of epiretinal membranes, complementing the structural information obtained with optical coherence tomography.

Hybrid In Silico/In Vitro Approaches for the Identification of Functional Cholesterol-Binding Domains in Membrane Proteins.

PubMed

Di Scala, Coralie; Fantini, Jacques

2017-01-01

In eukaryotic cells, cholesterol is an important regulator of a broad range of membrane proteins, including receptors, transporters, and ion channels. Understanding how cholesterol interacts with membrane proteins is a difficult task because structural data of these proteins complexed with cholesterol are scarce. Here, we describe a dual approach based on in silico studies of protein-cholesterol interactions, combined with physico-chemical measurements of protein insertion into cholesterol-containing monolayers. Our algorithm is validated through careful analysis of the effect of key mutations within and outside the predicted cholesterol-binding site. Our method is illustrated by a complete analysis of cholesterol-binding to Alzheimer's β-amyloid peptide, a protein that penetrates the plasma membrane of brain cells through a cholesterol-dependent process.

Chlamydiae interaction with the endoplasmic reticulum: contact, function and consequences.

PubMed

Derré, Isabelle

2015-07-01

Chlamydiae and chlamydiae-related organisms are obligate intracellular bacterial pathogens. They reside in a membrane-bound compartment termed the inclusion and have evolved sophisticated mechanisms to interact with cellular organelles. This review focuses on the nature, the function(s) and the consequences of chlamydiae-inclusion interaction with the endoplasmic reticulum (ER). The inclusion membrane establishes very close contact with the ER at specific sites termed ER-inclusion membrane contact sites (MCSs). These MCSs are constituted of a specific set of factors, including the C. trachomatis effector protein IncD and the host cell proteins CERT and VAPA/B. Because CERT and VAPA/B have a demonstrated role in the non-vesicular trafficking of lipids between the ER and the Golgi, it was proposed that Chlamydia establish MCSs with the ER to acquire host lipids. However, the recruitment of additional factors to ER-inclusion MCSs, such as the ER calcium sensor STIM1, may suggest additional functions unrelated to lipid acquisition. Finally, chlamydiae interaction with the ER appears to induce the ER stress response, but this response is quickly dampened by chlamydiae to promote host cell survival. © 2015 John Wiley & Sons Ltd.

Atorvastatin affects negatively respiratory function of isolated endothelial mitochondria.

PubMed

Broniarek, Izabela; Jarmuszkiewicz, Wieslawa

2018-01-01

The purpose of this research was to elucidate the direct effects of two popular blood cholesterol-lowering drugs used to treat cardiovascular diseases, atorvastatin and pravastatin, on respiratory function, membrane potential, and reactive oxygen species formation in mitochondria isolated from human umbilical vein endothelial cells (EA.hy926 cell line). Hydrophilic pravastatin did not significantly affect endothelial mitochondria function. In contrast, hydrophobic calcium-containing atorvastatin induced a loss of outer mitochondrial membrane integrity, an increase in hydrogen peroxide formation, and reductions in maximal (phosphorylating or uncoupled) respiratory rate, membrane potential and oxidative phosphorylation efficiency. The atorvastatin-induced changes indicate an impairment of mitochondrial function at the level of ATP synthesis and at the level of the respiratory chain, likely at complex I and complex III. The atorvastatin action on endothelial mitochondria was highly dependent on calcium ions and led to a disturbance in mitochondrial calcium homeostasis. Uptake of calcium ions included in atorvastatin molecule induced mitochondrial uncoupling that enhanced the inhibition of the mitochondrial respiratory chain by atorvastatin. Our results indicate that hydrophobic calcium-containing atorvastatin, widely used as anti-atherosclerotic agent, has a direct negative action on isolated endothelial mitochondria. Copyright © 2017. Published by Elsevier Inc.

Direct Capture of Functional Proteins from Mammalian Plasma Membranes into Nanodiscs.

PubMed

Roy, Jahnabi; Pondenis, Holly; Fan, Timothy M; Das, Aditi

2015-10-20

Mammalian plasma membrane proteins make up the largest class of drug targets yet are difficult to study in a cell free system because of their intransigent nature. Herein, we perform direct encapsulation of plasma membrane proteins derived from mammalian cells into a functional nanodisc library. Peptide fingerprinting was used to analyze the proteome of the incorporated proteins in nanodiscs and to further demonstrate that the lipid composition of the nanodiscs directly affects the class of protein that is incorporated. Furthermore, the functionality of the incorporated membrane proteome was evaluated by measuring the activity of membrane proteins: Na(+)/K(+)-ATPase and receptor tyrosine kinases. This work is the first report of the successful establishment and characterization of a cell free functional library of mammalian membrane proteins into nanodiscs.

Single-step electrochemical functionalization of double-walled carbon nanotube (DWCNT) membranes and the demonstration of ionic rectification

PubMed Central

2013-01-01

Carbon nanotube (CNT) membranes allow the mimicking of natural ion channels for applications in drug delivery and chemical separation. Double-walled carbon nanotube membranes were simply functionalized with dye in a single step instead of the previous two-step functionalization. Non-faradic electrochemical impedance spectra indicated that the functionalized gatekeeper by single-step modification can be actuated to mimic the protein channel under bias. This functional chemistry was proven by a highly efficient ion rectification, wherein the highest experimental rectification factor of ferricyanide was up to 14.4. One-step functionalization by electrooxidation of amine provides a simple and promising functionalization chemistry for the application of CNT membranes. PMID:23758999

Lipid bilayer thickness determines cholesterol's location in model membranes

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

Marquardt, Drew; Heberle, Frederick A.; Greathouse, Denise V.

Cholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature. Using a combination of neutron scattering and solid-state 2H NMR, we have determined the location and orientation of cholesterol in phosphatidylcholine (PC) model membranes having fatty acids of differentmore » lengths and degrees of unsaturation. The data establish that cholesterol reorients rapidly about the bilayer normal in all the membranes studied, but is tilted and forced to span the bilayer midplane in the very thin bilayers. The possibility that cholesterol lies flat in the middle of bilayers, including those made from PC lipids containing polyunsaturated fatty acids (PUFAs), is ruled out. Finally, these results support the notion that hydrophobic thickness is the primary determinant of cholesterol's location in membranes.« less

Lipid bilayer thickness determines cholesterol's location in model membranes

DOE PAGES

Marquardt, Drew; Heberle, Frederick A.; Greathouse, Denise V.; ...

2016-10-11

Cholesterol is an essential biomolecule of animal cell membranes, and an important precursor for the biosynthesis of certain hormones and vitamins. It is also thought to play a key role in cell signaling processes associated with functional plasma membrane microdomains (domains enriched in cholesterol), commonly referred to as rafts. In all of these diverse biological phenomena, the transverse location of cholesterol in the membrane is almost certainly an important structural feature. Using a combination of neutron scattering and solid-state 2H NMR, we have determined the location and orientation of cholesterol in phosphatidylcholine (PC) model membranes having fatty acids of differentmore » lengths and degrees of unsaturation. The data establish that cholesterol reorients rapidly about the bilayer normal in all the membranes studied, but is tilted and forced to span the bilayer midplane in the very thin bilayers. The possibility that cholesterol lies flat in the middle of bilayers, including those made from PC lipids containing polyunsaturated fatty acids (PUFAs), is ruled out. Finally, these results support the notion that hydrophobic thickness is the primary determinant of cholesterol's location in membranes.« less

Heat Stress Dictates Microbial Lipid Composition along a Thermal Gradient in Marine Sediments

PubMed Central

Sollich, Miriam; Yoshinaga, Marcos Y.; Häusler, Stefan; Price, Roy E.; Hinrichs, Kai-Uwe; Bühring, Solveig I.

2017-01-01

Temperature exerts a first-order control on microbial populations, which constantly adjust the fluidity and permeability of their cell membrane lipids to minimize loss of energy by ion diffusion across the membrane. Analytical advances in liquid chromatography coupled to mass spectrometry have allowed the detection of a stunning diversity of bacterial and archaeal lipids in extreme environments such as hot springs, hydrothermal vents and deep subsurface marine sediments. Here, we investigated a thermal gradient from 18 to 101°C across a marine sediment field and tested the hypothesis that cell membrane lipids provide a major biochemical basis for the bioenergetics of archaea and bacteria under heat stress. This paper features a detailed lipidomics approach with the focus on membrane lipid structure-function. Membrane lipids analyzed here include polar lipids of bacteria and polar and core lipids of archaea. Reflecting the low permeability of their ether-linked isoprenoids, we found that archaeal polar lipids generally dominate over bacterial lipids in deep layers of the sediments influenced by hydrothermal fluids. A close examination of archaeal and bacterial lipids revealed a membrane quandary: not only low permeability, but also increased fluidity of membranes are required as a unified property of microbial membranes for energy conservation under heat stress. For instance, bacterial fatty acids were composed of longer chain lengths in concert with higher degree of unsaturation while archaea modified their tetraethers by incorporation of additional methyl groups at elevated sediment temperatures. It is possible that these configurations toward a more fluidized membrane at elevated temperatures are counterbalanced by the high abundance of archaeal glycolipids and bacterial sphingolipids, which could reduce membrane permeability through strong intermolecular hydrogen bonding. Our results provide a new angle for interpreting membrane lipid structure-function enabling archaea and bacteria to survive and grow in hydrothermal systems. PMID:28878741

Membrane Protein Structure, Function, and Dynamics: a Perspective from Experiments and Theory

DOE PAGES

Cournia, Zoe; Allen, Toby W.; Andricioaei, Ioan; ...

2015-06-11

It is fundamental for the flourishing biological cells that membrane proteins mediate the process. Membrane-embedded transporters move ions and larger solutes across membranes; receptors mediate communication between the cell and its environment and membrane-embedded enzymes catalyze chemical reactions. Understanding these mechanisms of action requires knowledge of how the proteins couple to their fluid, hydrated lipid membrane environment. Here, we present here current studies in computational and experimental membrane protein biophysics, and show how they address outstanding challenges in understanding the complex environmental effects on the structure, function, and dynamics of membrane proteins.

Docosahexaenoic Acid and Cognition throughout the Lifespan

PubMed Central

Weiser, Michael J.; Butt, Christopher M.; Mohajeri, M. Hasan

2016-01-01

Docosahexaenoic acid (DHA) is the predominant omega-3 (n-3) polyunsaturated fatty acid (PUFA) found in the brain and can affect neurological function by modulating signal transduction pathways, neurotransmission, neurogenesis, myelination, membrane receptor function, synaptic plasticity, neuroinflammation, membrane integrity and membrane organization. DHA is rapidly accumulated in the brain during gestation and early infancy, and the availability of DHA via transfer from maternal stores impacts the degree of DHA incorporation into neural tissues. The consumption of DHA leads to many positive physiological and behavioral effects, including those on cognition. Advanced cognitive function is uniquely human, and the optimal development and aging of cognitive abilities has profound impacts on quality of life, productivity, and advancement of society in general. However, the modern diet typically lacks appreciable amounts of DHA. Therefore, in modern populations, maintaining optimal levels of DHA in the brain throughout the lifespan likely requires obtaining preformed DHA via dietary or supplemental sources. In this review, we examine the role of DHA in optimal cognition during development, adulthood, and aging with a focus on human evidence and putative mechanisms of action. PMID:26901223

Tubby family proteins are adapters for ciliary trafficking of integral membrane proteins

PubMed Central

Shimada, Issei S.; Loriot, Evan

2017-01-01

The primary cilium is a paradigmatic organelle for studying compartmentalized signaling; however, unlike soluble protein trafficking, processes targeting integral membrane proteins to cilia are poorly understood. In this study, we determine that the tubby family protein TULP3 functions as a general adapter for ciliary trafficking of structurally diverse integral membrane cargo, including multiple reported and novel rhodopsin family G protein–coupled receptors (GPCRs) and the polycystic kidney disease–causing polycystin 1/2 complex. The founding tubby family member TUB also localizes to cilia similar to TULP3 and determines trafficking of a subset of these GPCRs to neuronal cilia. Using minimal ciliary localization sequences from GPCRs and fibrocystin (also implicated in polycystic kidney disease), we demonstrate these motifs to be sufficient and TULP3 dependent for ciliary trafficking. We propose a three-step model for TULP3/TUB-mediated ciliary trafficking, including the capture of diverse membrane cargo by the tubby domain in a phosphoinositide 4,5-bisphosphate (PI(4,5)P2)-dependent manner, ciliary delivery by intraflagellar transport complex A binding to the TULP3/TUB N terminus, and subsequent release into PI(4,5)P2-deficient ciliary membrane. PMID:28154160

Structural elucidation of the interaction between neurodegenerative disease-related tau protein with model lipid membranes

NASA Astrophysics Data System (ADS)

Jones, Emmalee M.

A protein's sequence of amino acids determines how it folds. That folded structure is linked to protein function, and misfolding to dysfunction. Protein misfolding and aggregation into beta-sheet rich fibrillar aggregates is connected with over 20 neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized in part by misfolding, aggregation and deposition of the microtubule associated tau protein into neurofibrillary tangles (NFTs). However, two questions remain: What is tau's fibrillization mechanism, and what is tau's cytotoxicity mechanism? Tau is prone to heterogeneous interactions, including with lipid membranes. Lipids have been found in NFTs, anionic lipid vesicles induced aggregation of the microtubule binding domain of tau, and other protein aggregates induced ion permeability in cells. This evidence prompted our investigation of tau's interaction with model lipid membranes to elucidate the structural perturbations those interactions induced in tau protein and in the membrane. We show that although tau is highly charged and soluble, it is highly surface active and preferentially interacts with anionic membranes. To resolve molecular-scale structural details of tau and model membranes, we utilized X-ray and neutron scattering techniques. X-ray reflectivity indicated tau aggregated at air/water and anionic lipid membrane interfaces and penetrated into membranes. More significantly, membrane interfaces induced tau protein to partially adopt a more compact conformation with density similar to folded protein and ordered structure characteristic of beta-sheet formation. This suggests possible membrane-based mechanisms of tau aggregation. Membrane morphological changes were seen using fluorescence microscopy, and X-ray scattering techniques showed tau completely disrupts anionic membranes, suggesting an aggregate-based cytotoxicity mechanism. Further investigation of protein constructs and a "hyperphosphorylation" disease mimic helped clarify the role of the microtubule binding domain in anionic lipid affinity and demonstrated even "hyperphosphorylation" did not prevent interaction with anionic membranes. Additional studies investigated more complex membrane models to increase physiological relevance. These insights revealed structural changes in tau protein and lipid membranes after interaction. We observed tau's affinity for interfaces, and aggregation and compaction once tau partitions to interfaces. We observed the beginnings of beta-sheet formation in tau at anionic lipid membranes. We also examined disruption to the membrane on a molecular scale.

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

Membrane Stored Curvature Elastic Stress Modulates Recruitment of Maintenance Proteins PspA and Vipp1.

PubMed

McDonald, Christopher; Jovanovic, Goran; Ces, Oscar; Buck, Martin

2015-09-01

Phage shock protein A (PspA), which is responsible for maintaining inner membrane integrity under stress in enterobacteria, and vesicle-inducting protein in plastids 1 (Vipp1), which functions for membrane maintenance and thylakoid biogenesis in cyanobacteria and plants, are similar peripheral membrane-binding proteins. Their homologous N-terminal amphipathic helices are required for membrane binding; however, the membrane features recognized and required for expressing their functionalities have remained largely uncharacterized. Rigorously controlled, in vitro methodologies with lipid vesicles and purified proteins were used in this study and provided the first biochemical and biophysical characterizations of membrane binding by PspA and Vipp1. Both proteins are found to sense stored curvature elastic (SCE) stress and anionic lipids within the membrane. PspA has an enhanced sensitivity for SCE stress and a higher affinity for the membrane than Vipp1. These variations in binding may be crucial for some of the proteins' differing roles in vivo. Assays probing the transcriptional regulatory function of PspA in the presence of vesicles showed that a relief of transcription inhibition occurs in an SCE stress-specific manner. This in vitro recapitulation of membrane stress-dependent transcription control suggests that the Psp response may be mounted in vivo when a cell's inner membrane experiences increased SCE stress. All cell types maintain the integrity of their membrane systems. One widely distributed membrane stress response system in bacteria is the phage shock protein (Psp) system. The central component, peripheral membrane protein PspA, which mitigates inner membrane stress in bacteria, has a counterpart, Vipp1, which functions for membrane maintenance and thylakoid biogenesis in plants and photosynthetic bacteria. Membrane association of both these proteins is accepted as playing a pivotal role in their functions. Here we show that direct membrane binding by PspA and Vipp1 is driven by two physio-chemical signals, one of which is membrane stress specific. Our work points to alleviation of membrane stored curvature elastic stress by amphipathic helix insertions as an attractive mechanism for membrane maintenance by PspA and Vipp1. Furthermore, the identification of a physical, stress-related membrane signal suggests a unilateral mechanism that promotes both binding of PspA and induction of the Psp response. Copyright © 2015 McDonald et al.

Aquaporins in Clinical Medicine

PubMed Central

Verkman, A.S.

2012-01-01

The aquaporins are a family of membrane water channels, some of which also transport glycerol. They are involved in a wide range of physiological functions (including water/salt homeostasis, exocrine fluid secretion, and epidermal hydration) and human diseases (including glaucoma, cancer, epilepsy, and obesity). At the cellular level, aquaporin-mediated osmotic water transport across cell plasma membranes facilitates transepithelial fluid transport, cell migration, and neuroexcitation; aquaporin-mediated glycerol transport regulates cell proliferation, adipocyte metabolism, and epidermal water retention. Genetic diseases caused by loss-of-function mutations in aquaporins include nephrogenic diabetes insipidus and congenital cataracts. The neuroinflammatory demyelinating disease neuromyelitis optica is marked by pathogenic autoantibodies against astrocyte water channel aquaporin-4. There remain broad opportunities for the development of aquaporin-based diagnostics and therapeutics. Disease-relevant aquaporin polymorphisms are beginning to be explored. There is great promise in the development of small-molecule aquaporin modulators for therapy of some types of refractory edema, brain swelling, neuroinflammation, glaucoma, epilepsy, cancer, pain, and obesity. PMID:22248325

Nanoscale patterning controls inorganic-membrane interface structure

NASA Astrophysics Data System (ADS)

Almquist, Benjamin D.; Verma, Piyush; Cai, Wei; Melosh, Nicholas A.

2011-02-01

The ability to non-destructively integrate inorganic structures into or through biological membranes is essential to realizing full bio-inorganic integration, including arrayed on-chip patch-clamps, drug delivery, and biosensors. Here we explore the role of nanoscale patterning on the strength of biomembrane-inorganic interfaces. AFM measurements show that inorganic probes functionalized with hydrophobic bands with thicknesses complimentary to the hydrophobic lipid bilayer core exhibit strong attachment in the bilayer. As hydrophobic band thickness increases to 2-3 times the bilayer core the interfacial strength decreases, comparable to homogeneously hydrophobic probes. Analytical calculations and molecular dynamics simulations predict a transition between a `fused' interface and a `T-junction' that matches the experimental results, showing lipid disorder and defect formation for thicker bands. These results show that matching biological length scales leads to more intimate bio-inorganic junctions, enabling rational design of non-destructive membrane interfaces.The ability to non-destructively integrate inorganic structures into or through biological membranes is essential to realizing full bio-inorganic integration, including arrayed on-chip patch-clamps, drug delivery, and biosensors. Here we explore the role of nanoscale patterning on the strength of biomembrane-inorganic interfaces. AFM measurements show that inorganic probes functionalized with hydrophobic bands with thicknesses complimentary to the hydrophobic lipid bilayer core exhibit strong attachment in the bilayer. As hydrophobic band thickness increases to 2-3 times the bilayer core the interfacial strength decreases, comparable to homogeneously hydrophobic probes. Analytical calculations and molecular dynamics simulations predict a transition between a `fused' interface and a `T-junction' that matches the experimental results, showing lipid disorder and defect formation for thicker bands. These results show that matching biological length scales leads to more intimate bio-inorganic junctions, enabling rational design of non-destructive membrane interfaces. Electronic supplementary information (ESI) available: Breakthrough rate as a function of force plots for 5 nm, 10 nm and ∞-probes.. See DOI: 10.1039/c0nr00486c

Clinical features, course and prognosis of idiopathic membranous nephropathy depending on the presence of antibodies against M-type phospholipase A2 receptor.

PubMed

Jatem Escalante, Elías; Segarra Medrano, Alfons; Carnicer Cáceres, Clara; Martín-Gómez, M Adoración; Salcedo Allende, María Teresa; Ostos Roldan, Helena; Agraz Pamplona, Irene

2015-01-01

In membranous nephropathy, the presence of antibodies against M-type phospholipase A2 receptor is considered highly specific for idiopathic forms. However, no specific association to a particular clinical profile has been found for such antibodies. To assess potential differences in initial clinical profile, course and prognosis of idiopathic membranous nephropathy depending on the presence of anti-PLA2R antibodies. Eighty-five patients with idiopathic membranous nephropathy were included (55 anti-PLA2R-positive and 30 anti-PLA2R-negative). Clinical, biochemical and pathological variables were recorded at the time of diagnosis. Frequency of spontaneous remission, incidence of response to first-line therapy, frequency and number of recurrences, survival of renal function free from renal replacement therapy, survival of renal function free from chronic renal insufficiency and frequency of occurrence of malignant, infectious or autoimmune diseases during follow-up were recorded. At the time of diagnosis, anti-PLA2R-negative patients were significantly older and had a higher frequency of spontaneous remission. No differences were noted in the response to first-line treatment, frequency and number of recurrences, survival of renal function free from renal replacement therapy, or survival of renal function free from chronic renal insufficiency. Anti-PLA2R-negative patients with idiopathic membranous nephropathy were older and experienced spontaneous remission more often than anti-PLA2R-positive patients. No differences in terms of treatment response, recurrences, and final prognosis were observed between both groups of patients. Copyright © 2015 The Authors. Published by Elsevier España, S.L.U. All rights reserved.

Elucidation of the Block to Herpes Simplex Virus Egress in the Absence of Tegument Protein UL16 Reveals a Novel Interaction with VP22

PubMed Central

Starkey, Jason L.; Han, Jun; Chadha, Pooja; Marsh, Jacob A.

2014-01-01

UL16 is a tegument protein of herpes simplex virus (HSV) that is conserved among all members of the Herpesviridae, but its function is poorly understood. Previous studies revealed that UL16 is associated with capsids in the cytoplasm and interacts with the membrane protein UL11, which suggested a “bridging” function during cytoplasmic envelopment, but this conjecture has not been tested. To gain further insight, cells infected with UL16-null mutants were examined by electron microscopy. No defects in the transport of capsids to cytoplasmic membranes were observed, but the wrapping of capsids with membranes was delayed. Moreover, clusters of cytoplasmic capsids were often observed, but only near membranes, where they were wrapped to produce multiple capsids within a single envelope. Normal virion production was restored when UL16 was expressed either by complementing cells or from a novel position in the HSV genome. When the composition of the UL16-null viruses was analyzed, a reduction in the packaging of glycoprotein E (gE) was observed, which was not surprising, since it has been reported that UL16 interacts with this glycoprotein. However, levels of the tegument protein VP22 were also dramatically reduced in virions, even though this gE-binding protein has been shown not to depend on its membrane partner for packaging. Cotransfection experiments revealed that UL16 and VP22 can interact in the absence of other viral proteins. These results extend the UL16 interaction network beyond its previously identified binding partners to include VP22 and provide evidence that UL16 plays an important function at the membrane during virion production. PMID:24131716

Disordered Cold Regulated15 Proteins Protect Chloroplast Membranes during Freezing through Binding and Folding, But Do Not Stabilize Chloroplast Enzymes in Vivo1[W][OPEN

PubMed Central

Thalhammer, Anja; Bryant, Gary; Sulpice, Ronan; Hincha, Dirk K.

2014-01-01

Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low nonfreezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold-regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis (Arabidopsis thaliana). They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins. PMID:25096979

Relationship between CYP1A2 Localization and Lipid Microdomain Formation as a Function of Lipid Composition

PubMed Central

Brignac-Huber, Lauren M.; Reed, James R.; Eyer, Marilyn K.

2013-01-01

Cytochrome P450 (P450) function requires the interaction of P450 and NADPH-cytochrome P450 reductase (CPR) in membranes, and is frequently studied using reconstituted systems composed solely of phosphatidylcholine. There is increasing evidence that other endoplasmic reticulum (ER) lipids can affect P450 structure, activity, and interactions with CPR. Some of these lipid effects have been attributed to the formation of organized liquid-ordered (lo) domains. The goal of this study was to determine if lo domains were formed in P450 reconstituted systems mimicking the ER membrane. CYP1A2, when incorporated in “ER-like” lipid vesicles, displayed detergent insolubility after treatment with Brij 98 and centrifugation in a sucrose gradient. Lipid probes were employed to identify domain formation in both ER-like vesicles and model membranes known to form lo domains. Changes in fluorescence resonance energy transfer (FRET) using an established donor/acceptor FRET pair in both ER-like and model lo-forming systems demonstrated the coexistence of lo- and liquid-disordered domains as a function of cholesterol and sphingomyelin content. Similarly, 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), a probe that reports on membrane organization, showed that cholesterol and sphingomyelin increased membrane order. Finally, brominated-phosphatidylcholine allowed for monitoring of the location of both CPR and CYP1A2 within the lo regions of ER-like systems. Taken together, the results demonstrate that ER-like vesicles generate microdomains, and both CYP1A2 and CPR predominantly localize into lo membrane regions. Probe fluorescent responses suggest that lipid microdomains form in these vesicles whether or not enzymes are included in the reconstituted systems. Thus, it does not appear that the proteins are critical for stabilizing lo domains. PMID:23963955

From Embryonic Development to Human Diseases: The Functional Role of Caveolae/Caveolin

PubMed Central

Sohn, Jihee; Brick, Rachel M.; Tuan, Rocky S.

2017-01-01

Caveolae, an almost ubiquitous, structural component of the plasma membrane, play a critical role in many functions essential for proper cell function, including membrane trafficking, signal transduction, extracellular matrix remodeling, and tissue regeneration. Three main types of caveolin proteins have been identified from caveolae since the discovery of caveolin-1 in the early 1990s. All three (Cav-1, Cav-2, and Cav-3) play crucial roles in mammalian physiology, and can effect pathogenesis in a wide range of human diseases. While many biological activities of caveolins have been uncovered since its discovery, their role and regulation in embryonic develop remain largely poorly understood, although there is increasing evidence that caveolins may be linked to lung and brain birth defects. Further investigations are clearly needed to decipher how caveolae/caveolins mediate cellular functions and activities of normal embryogenesis and how their perturbations contribute to developmental disorders. PMID:26991990

FERMT2 links cortical actin structures, plasma membrane tension and focal adhesion function to stabilize podocyte morphology.

PubMed

Yasuda-Yamahara, M; Rogg, M; Frimmel, J; Trachte, P; Helmstaedter, M; Schroder, P; Schiffer, M; Schell, C; Huber, T B

2018-01-11

Simplification and retraction of podocyte protrusions, generally termed as foot process effacement, is a uniform pathological pattern observed in the majority of glomerular disease, including focal segmental glomerulosclerosis. However, it is still incompletely understood how the interaction of cortical actin structures, actomyosin contractility and focal adhesions, is being orchestrated to control foot process morphology in health and disease. By uncovering the functional role of fermitin family member 2 (FERMT2 or kindlin-2) in podocytes, we provide now evidence, how cell-extracellular matrix (ECM) interactions modulate membrane tension and actomyosin contractility. A genetic modeling approach was applied by deleting FERMT2 in a set of in vivo systems as well as in CRISPR/Cas9 modified human podocytes. Loss of FERMT2 results in altered cortical actin composition, cell cortex destabilization associated with plasma membrane blebbing and a remodeling of focal adhesions. We further show that FERMT2 knockout podocytes have high levels of RhoA activation and concomitantly increased actomyosin contractility. Inhibition of actomyosin tension reverses the membrane blebbing phenotype. Thus, our findings establish a direct link between cell-matrix adhesions, cortical actin structures and plasma membrane tension allowing to better explain cell morphological changes in foot process effacement. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Protein aggregation induced during glass bead lysis of yeast

PubMed Central

Papanayotou, Irene; Sun, Beimeng; Roth, Amy F.; Davis, Nicholas G.

2013-01-01

Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein–protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation. PMID:20641011

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function

PubMed Central

Simpson, Brent W.; Owens, Tristan W.; Orabella, Matthew J.; Davis, Rebecca M.; May, Janine M.; Trauger, Sunia A.

2016-01-01

ABSTRACT The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB2FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB2FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. PMID:27795402

Immunolocalization of aquaporin CHIP in the guinea pig inner ear.

PubMed

Stanković, K M; Adams, J C; Brown, D

1995-12-01

Aquaporin CHIP (AQP-CHIP) is a water channel protein previously identified in red blood cells and water transporting epithelia. The inner ear is an organ of hearing and balance whose normal function depends critically on maintenance of fluid homeostasis. In this study, AQP-CHIP, or a close homologue, was found in specific cells of the inner ear, as assessed by immunocytochemistry with the use of affinity-purified polyclonal antibodies against AQP-CHIP.AQP-CHIP was predominantly found in fibrocytes in close association with bone, including most of the cells lining the bony labyrinth and in fibrocytes lining the endolymphatic duct and sac. AQP-CHIP-positive cells not directly apposing bone include cells under the basilar membrane, some type III fibrocytes of the spiral ligament, fibrocytes of the spiral limbus, and the trabecular perilymphatic tissue extending from the membranous to the bony labyrinth. AQP-CHIP was also found in the periosteum of the middle ear and cranial bones, as well as in chondrocytes of the oval window and stapes. The distribution of AQP-CHIP in the inner ear suggests that AQP-CHIP may have special significance for maintenance of bone and the basilar membrane, and for function of the spiral ligament.

Cyclic and Linear Monoterpenes in Phospholipid Membranes: Phase Behavior, Bilayer Structure, and Molecular Dynamics.

PubMed

Pham, Quoc Dat; Topgaard, Daniel; Sparr, Emma

2015-10-13

Monoterpenes are abundant in essential oils extracted from plants. These relatively small and hydrophobic molecules have shown important biological functions, including antimicrobial activity and membrane penetration enhancement. The interaction between the monoterpenes and lipid bilayers is considered important to the understanding of the biological functions of monoterpenes. In this study, we investigated the effect of cyclic and linear monoterpenes on the structure and dynamics of lipids in model membranes. We have studied the ternary system 1,2-dimyristoyl-sn-glycero-3-phosphocholine-monoterpene-water as a model with a focus on dehydrated conditions. By combining complementary techniques, including differential scanning calorimetry, solid-state nuclear magnetic resonance, and small- and wide-angle X-ray scattering, bilayer structure, phase transitions, and lipid molecular dynamics were investigated at different water contents. Monoterpenes cause pronounced melting point depression and phase segregation in lipid bilayers, and the extent of these effects depends on the hydration conditions. The addition of a small amount of thymol to the fluid bilayer (volume fraction of 0.03 in the bilayer) leads to an increased order in the acyl chain close to the bilayer interface. The findings are discussed in relation to biological systems and lipid formulations.

Membrane of Functionalized Reduced Graphene Oxide Nanoplates with Angstrom-Level Channels

PubMed Central

Lee, Byeongho; Li, Kunzhou; Yoon, Hong Sik; Yoon, Jeyong; Mok, Yeongbong; Lee, Yan; Lee, Hong H.; Kim, Yong Hyup

2016-01-01

Membranes with atomic level pores or constrictions are valuable for separation and catalysis. We report a graphene-based membrane with an interlayer spacing of 3.7 angstrom (Å). When graphene oxide nanoplates are functionalized and then reduced, the laminated reduced graphene oxide (rGO) nanoplates or functionalized rGO membrane is little affected by an intercalated fluid, and the interlayer spacing of 3.7 Å increases only to 4.4 Å in wetted state, in contrast to the graphene oxide (GO) membrane whose interlayer spacing increases from 9 Å to 13 Å in wetted state. When applied to ion separation, this membrane reduced the permeation rate of small ions such as K+ and Na+ by three orders of magnitude compared to the GO membrane. PMID:27306853

FIG4 regulates lysosome membrane homeostasis independent of phosphatase function.

PubMed

Bharadwaj, Rajnish; Cunningham, Kathleen M; Zhang, Ke; Lloyd, Thomas E

2016-02-15

FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

FIG4 regulates lysosome membrane homeostasis independent of phosphatase function

PubMed Central

Bharadwaj, Rajnish; Cunningham, Kathleen M.; Zhang, Ke; Lloyd, Thomas E.

2016-01-01

FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS. PMID:26662798

Caenorhabditis elegans as a model to study renal development and disease: sexy cilia.

PubMed

Barr, Maureen M

2005-02-01

The nematode Caenorhabditis elegans has no kidney per se, yet "the worm" has proved to be an excellent model to study renal-related issues, including tubulogenesis of the excretory canal, membrane transport and ion channel function, and human genetic diseases including autosomal dominant polycystic kidney disease (ADPKD). The goal of this review is to explain how C. elegans has provided insight into cilia development, cilia function, and human cystic kidney diseases.

The Multifaceted Role of SNARE Proteins in Membrane Fusion

PubMed Central

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

2017-01-01

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

The Multifaceted Role of SNARE Proteins in Membrane Fusion.

PubMed

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

2017-01-01

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

Monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in Streptococcus pneumoniae using fluorescent dyes.

PubMed

Clementi, Emily A; Marks, Laura R; Roche-Håkansson, Hazeline; Håkansson, Anders P

2014-02-17

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca(2+), K(+), and H(+), respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

PubMed

Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

2011-11-22

Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41. © 2011 American Chemical Society

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion

NASA Astrophysics Data System (ADS)

Dong, Jinlan; Bruening, Merlin L.

2015-07-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO2 nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Functionalizing Microporous Membranes for Protein Purification and Protein Digestion.

PubMed

Dong, Jinlan; Bruening, Merlin L

2015-01-01

This review examines advances in the functionalization of microporous membranes for protein purification and the development of protease-containing membranes for controlled protein digestion prior to mass spectrometry analysis. Recent studies confirm that membranes are superior to bead-based columns for rapid protein capture, presumably because convective mass transport in membrane pores rapidly brings proteins to binding sites. Modification of porous membranes with functional polymeric films or TiO₂ nanoparticles yields materials that selectively capture species ranging from phosphopeptides to His-tagged proteins, and protein-binding capacities often exceed those of commercial beads. Thin membranes also provide a convenient framework for creating enzyme-containing reactors that afford control over residence times. With millisecond residence times, reactors with immobilized proteases limit protein digestion to increase sequence coverage in mass spectrometry analysis and facilitate elucidation of protein structures. This review emphasizes the advantages of membrane-based techniques and concludes with some challenges for their practical application.

Nanomaterial interactions with biomembranes: Bridging the gap between soft matter models and biological context.

PubMed

Werner, Marco; Auth, Thorsten; Beales, Paul A; Fleury, Jean Baptiste; Höök, Fredrik; Kress, Holger; Van Lehn, Reid C; Müller, Marcus; Petrov, Eugene P; Sarkisov, Lev; Sommer, Jens-Uwe; Baulin, Vladimir A

2018-04-03

Synthetic polymers, nanoparticles, and carbon-based materials have great potential in applications including drug delivery, gene transfection, in vitro and in vivo imaging, and the alteration of biological function. Nature and humans use different design strategies to create nanomaterials: biological objects have emerged from billions of years of evolution and from adaptation to their environment resulting in high levels of structural complexity; in contrast, synthetic nanomaterials result from minimalistic but controlled design options limited by the authors' current understanding of the biological world. This conceptual mismatch makes it challenging to create synthetic nanomaterials that possess desired functions in biological media. In many biologically relevant applications, nanomaterials must enter the cell interior to perform their functions. An essential transport barrier is the cell-protecting plasma membrane and hence the understanding of its interaction with nanomaterials is a fundamental task in biotechnology. The authors present open questions in the field of nanomaterial interactions with biological membranes, including: how physical mechanisms and molecular forces acting at the nanoscale restrict or inspire design options; which levels of complexity to include next in computational and experimental models to describe how nanomaterials cross barriers via passive or active processes; and how the biological media and protein corona interfere with nanomaterial functionality. In this Perspective, the authors address these questions with the aim of offering guidelines for the development of next-generation nanomaterials that function in biological media.

Multiplexed biomimetic lipid membranes on graphene by dip-pen nanolithography

PubMed Central

Hirtz, Michael; Oikonomou, Antonios; Georgiou, Thanasis; Fuchs, Harald; Vijayaraghavan, Aravind

2013-01-01

The application of graphene in sensor devices depends on the ability to appropriately functionalize the pristine graphene. Here we show the direct writing of tailored phospholipid membranes on graphene using dip-pen nanolithography. Phospholipids exhibit higher mobility on graphene compared with the commonly used silicon dioxide substrate, leading to well-spread uniform membranes. Dip-pen nanolithography allows for multiplexed assembly of phospholipid membranes of different functionalities in close proximity to each other. The membranes are stable in aqueous environments and we observe electronic doping of graphene by charged phospholipids. On the basis of these results, we propose phospholipid membranes as a route for non-covalent immobilization of various functional groups on graphene for applications in biosensing and biocatalysis. As a proof of principle, we demonstrate the specific binding of streptavidin to biotin-functionalized membranes. The combination of atomic force microscopy and binding experiments yields a consistent model for the layer organization within phospholipid stacks on graphene. PMID:24107937

Mechanisms of sterol uptake and transport in yeast.

PubMed

Jacquier, Nicolas; Schneiter, Roger

2012-03-01

Sterols are essential lipid components of eukaryotic membranes. Here we summarize recent advances in understanding how sterols are transported between different membranes. Baker's yeast is a particularly attractive organism to dissect this lipid transport pathway, because cells can synthesize their own major sterol, ergosterol, in the membrane of the endoplasmic reticulum from where it is then transported to the plasma membrane. However, Saccharomyces cerevisiae is also a facultative anaerobic organism, which becomes sterol auxotroph in the absence of oxygen. Under these conditions, cells take up sterol from the environment and transport the lipid back into the membrane of the endoplasmic reticulum, where the free sterol becomes esterified and is then stored in lipid droplets. Steryl ester formation is thus a reliable readout to assess the back-transport of exogenously provided sterols from the plasma membrane to the endoplasmic reticulum. Structure/function analysis has revealed that the bulk membrane function of the fungal ergosterol can be provided by structurally related sterols, including the mammalian cholesterol. Foreign sterols, however, are subject to a lipid quality control cycle in which the sterol is reversibly acetylated. Because acetylated sterols are efficiently excreted from cells, the substrate specificity of the deacetylating enzymes determines which sterols are retained. Membrane-bound acetylated sterols are excreted by the secretory pathway, more soluble acetylated sterol derivatives such as the steroid precursor pregnenolone, on the other hand, are excreted by a pathway that is independent of vesicle formation and fusion. Further analysis of this lipid quality control cycle is likely to reveal novel insight into the mechanisms that ensure sterol homeostasis in eukaryotic cells. Article from a special issue on Steroids and Microorganisms. Copyright © 2010. Published by Elsevier Ltd.

The Xylella fastidiosa PD1063 Protein Is Secreted in Association with Outer Membrane Vesicles

PubMed Central

Pierce, Brittany K.; Voegel, Tanja; Kirkpatrick, Bruce C.

2014-01-01

Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa. PMID:25426629

The Xylella fastidiosa PD1063 protein is secreted in association with outer membrane vesicles.

PubMed

Pierce, Brittany K; Voegel, Tanja; Kirkpatrick, Bruce C

2014-01-01

Xylella fastidiosa is a gram-negative, xylem-limited plant pathogenic bacterium that causes disease in a variety of economically important agricultural crops including Pierce's disease of grapevines. Xylella fastidiosa biofilms formed in the xylem vessels of plants play a key role in early colonization and pathogenicity by providing a protected niche and enhanced cell survival. Here we investigate the role of Xylella fastidiosa PD1063, the predicted ortholog of Xanthomonas oryzae pv. oryzae PXO_03968, which encodes an outer membrane protein. To assess the function of the Xylella fastidiosa ortholog, we created Xylella fastidiosa mutants deleted for PD1063 and then assessed biofilm formation, cell-cell aggregation and cell growth in vitro. We also assessed disease severity and pathogen titers in grapevines mechanically inoculated with the Xylella fastidiosa PD1063 mutant. We found a significant decrease in cell-cell aggregation among PD1063 mutants but no differences in cell growth, biofilm formation, disease severity or titers in planta. Based on the demonstration that Xanthomonas oryzae pv. oryzae PXO_03968 encodes an outer membrane protein, secreted in association with outer membrane vesicles, we predicted that PD1063 would also be secreted in a similar manner. Using anti-PD1063 antibodies, we found PD1063 in the supernatant and secreted in association with outer membrane vesicles. PD1063 purified from the supernatant, outer membrane fractions and outer membrane vesicles was 19.2 kD, corresponding to the predicted size of the processed protein. Our findings suggest Xylella fastidiosa PD1063 is not essential for development of Pierce's disease in Vitis vinifera grapevines although further research is required to determine the function of the PD1063 outer membrane protein in Xylella fastidiosa.

Disruption of Ankyrin B and Caveolin-1 Interaction Sites Alters Na+,K+-ATPase Membrane Diffusion.

PubMed

Junghans, Cornelia; Vukojević, Vladana; Tavraz, Neslihan N; Maksimov, Eugene G; Zuschratter, Werner; Schmitt, Franz-Josef; Friedrich, Thomas

2017-11-21

The Na + ,K + -ATPase is a plasma membrane ion transporter of high physiological importance for ion homeostasis and cellular excitability in electrically active tissues. Mutations in the genes coding for Na + ,K + -ATPase α-subunit isoforms lead to severe human pathologies including Familial Hemiplegic Migraine type 2, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia Parkinsonism, or epilepsy. Many of the reported mutations lead to change- or loss-of-function effects, whereas others do not alter the functional properties, but lead to, e.g., reduced protein stability, reduced protein expression, or defective plasma membrane targeting. Na + ,K + -ATPase frequently assembles with other membrane transporters or cellular matrix proteins in specialized plasma membrane microdomains, but the effects of these interactions on targeting or protein mobility are elusive so far. Mutation of established interaction motifs of the Na + ,K + -ATPase with ankyrin B and caveolin-1 are expected to result in changes in plasma membrane targeting, changes of the localization pattern, and of the diffusion behavior of the enzyme. We studied the consequences of mutations in these binding sites by monitoring diffusion of eGFP-labeled Na + ,K + -ATPase constructs in the plasma membrane of HEK293T cells by fluorescence correlation spectroscopy as well as fluorescence recovery after photobleaching or photoswitching, and observed significant differences compared to the wild-type enzyme, with synergistic effects for combinations of interaction site mutations. These measurements expand the possibilities to study the consequences of Na + ,K + -ATPase mutations and provide information about the interaction of Na + ,K + -ATPase α-isoforms with cellular matrix proteins, the cytoskeleton, or other membrane protein complexes. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Molecular Modeling of Lipid Aggregates: Theory and Application

NASA Astrophysics Data System (ADS)

Fenner, Joel Stewart

The ability of cell membranes to perform a wide variety of biological functions stems from the organization and composition of its molecular constituents. There are many engineering applications, such as liposome drug delivery carriers, whose functionality takes advantage of the structure to function relationship of lipid membranes. The fundamental understanding of the relationship between the thermodynamic behavior and structure of lipid membranes and the molecular properties of their lipid constituents is crucial to the successful design of lipid related applications. However, information about how the local microscopic composition of lipid membranes responds to the presence of proteins and nanomaterials is challenging given the intrinsic experimental and theoretical difficulties of studying such small-scale systems. The present work generalizes a self consistent mean field theory for the study of the thermodynamic and structural behavior of lipid bilayers as a function of its molecular composition and physicochemical environments. This novel molecular theory provides with the ability of performing systematic thermodynamic calculations at relatively low computational costs while considering a detailed molecular description of the system under study. The competition of all relevant molecular interactions, such as electrostatics, vdW and chemical equilibria, in the membrane system is described. The developed molecular theory is applied to study how the protonation state of pH-sensitive amphiphiles in a membrane system affects the membrane's morphology. The molecular theory results demonstrate that the protonation state of ionizable groups within amphiphilic membranes shows a highly complex non-monotonic dependence on bulk salt concentration and pH strength. This result suggests that information about the pKa of the molecules is not sufficient to predict the protonation state of the ionizable groups in the membrane system. The molecular theory is also applied to study how the presence of proteins or functionalized nanoparticles near a multicomponent membrane surface leads to changes in its local membrane composition. The results support an electrostatic dependent recruitment mechanism of oncogenic RhoA proteins to the cell membrane. Finally, the molecular theory results describe how nanoparticle functionality and/or membrane molecular composition can be tuned to enhance or suppress nanoparticle adsorption on to phospholipid membranes.

Rhomboid intramembrane protease RHBDL4 triggers ER-export and non-canonical secretion of membrane-anchored TGFα

PubMed Central

Wunderle, Lina; Knopf, Julia D.; Kühnle, Nathalie; Morlé, Aymeric; Hehn, Beate; Adrain, Colin; Strisovsky, Kvido; Freeman, Matthew; Lemberg, Marius K.

2016-01-01

Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes. PMID:27264103

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

Fabrication and characterization of vertically aligned carbon-nanotube membranes

NASA Astrophysics Data System (ADS)

Castellano, Richard; Akin, Cevat; Purri, Matt; Shan, Jerry; Kim, Sangil; Fornasiero, Francesco

2015-11-01

Membranes having vertically-aligned carbon-nanotube (VACNT) pores offer promise as highly efficient and permeable membranes for use as breathable thin films, or in filtration and separation applications, among others. However, current membrane-fabrication techniques utilizing chemical-vapor-deposition-grown VACNT arrays are costly and difficult to scale up. We have developed a solution-based, electric-field-assisted approach as a cost-effective and scalable method to produce large-area VACNT membranes. Nanotubes are dispersed in a liquid polymer, and aligned and electrodeposited with the aid of an electric field prior to crosslinking the polymer to create VACNT membranes. We experimentally examine the electrodeposition process, focusing on parameters including the electric field, composition of the solution, and CNT functionalization that can affect the nanotube number density in the resulting membrane. We characterize the CNT pore size and number density and investigate the transport properties of the membrane. Size-exclusion tests are used to check for defects and infer the pore size of the VACNT membranes. Dry-gas membrane permeability is measured with a pressurized nitrogen-flow system, while moisture-vapor-transfer rate is measured with the ASTM-E96 upright-cup test. We discuss the measured transport properties of the solution-based, electric-field-fabricated VACNT membranes in reference to their application as breathable thin films. We would like to acknowledge DTRA for their funding and support of our research.

Introducing multiple bio-functional groups on the poly(ether sulfone) membrane substrate to fabricate an effective antithrombotic bio-interface.

PubMed

Wang, Lingren; He, Min; Gong, Tao; Zhang, Xiang; Zhang, Lincai; Liu, Tao; Ye, Wei; Pan, Changjiang; Zhao, Changsheng

2017-11-21

It has been widely recognized that functional groups on biomaterial surfaces play important roles in blood compatibility. To construct an effective antithrombotic bio-interface onto the poly(ether sulfone) (PES) membrane surface, bio-functional groups of sodium carboxylic (-COONa), sodium sulfonic (-SO 3 Na) and amino (-NH 2 ) groups were introduced onto the PES membrane surface in three steps: the synthesis of PES with carboxylic (-COOH) groups (CPES) and water-soluble PES with sodium sulfonic (-SO 3 Na) groups and amino (-NH 2 ) groups (SNPES); the introduction of carboxylic groups onto the PES membrane by blending CPES with PES; and the grafting of SNPES onto CPES/PES membranes via the coupling of amino groups and carboxyl groups. The physical/chemical properties and bioactivities were dependent on the proportions of the additives. After introducing bio-functional groups, the excellent hemocompatibility of the modified membranes was confirmed by the inhibited platelet adhesion and activation, prolonged clotting times, suppressed blood-related complement and leukocyte-related complement receptor activations. Furthermore, cell tests indicated that the modified membranes showed better cytocompatibility in endothelial cell proliferation than the pristine PES membrane due to the synergistic promotion of the functional groups. To sum up, these results suggested that modified membranes present great potential in fields using blood-contacting materials, such as hemodialysis and surface endothelialization.

Naphthenic acids removal from high TDS produced water by persulfate mediated iron oxide functionalized catalytic membrane, and by nanofiltration.

PubMed

Aher, Ashish; Papp, Joseph; Colburn, Andrew; Wan, Hongyi; Hatakeyama, Evan; Prakash, Prakhar; Weaver, Ben; Bhattacharyya, Dibakar

2017-11-01

Oil industries generate large amounts of produced water containing organic contaminants, such as naphthenic acids (NA) and very high concentrations of inorganic salts. Recovery of potable water from produced water can be highly energy intensive is some cases due to its high salt concentration, and safe discharge is more suitable. Here, we explored catalytic properties of iron oxide (Fe x O y nanoparticles) functionalized membranes in oxidizing NA from water containing high concentrations of total dissolved solids (TDS) using persulfate as an oxidizing agent. Catalytic decomposition of persulfate by Fe x O y functionalized membranes followed pseudo-first order kinetics with an apparent activation energy of 18 Kcal/mol. Fe x O y functionalized membranes were capable of lowering the NA concentrations to less than discharge limits of 10 ppm at 40 °C. Oxidation state of iron during reaction was quantified. Membrane performance was investigated for extended period of time. A coupled process of advanced oxidation catalyzed by membrane and nanofiltration was also evaluated. Commercially available nanofiltration membranes were found capable of retaining NA from water containing high concentrations of dissolved salts. Commercial NF membranes, Dow NF270 (Dow), and NF8 (Nanostone) had NA rejection of 79% and 82%, respectively. Retentate for the nanofiltration was further treated with advanced oxidation catalyzed by Fe x O y functionalized membrane for removal of NA.

Polymethylmethacrylate membrane with a series of serendipity.

PubMed

Sakai, Yoshitada

2011-01-01

Forty years have passed since the polymethylmethacrylate (PMMA) membrane was first developed. This article reviews its history and explains its longevity. The membrane was developed through application of a stereocomplex phenomenon that is observed upon mixture of isotactic and syndiotactic PMMA polymers. Filtryzer(TM) B1 and B2 were approved in Japan in 1977. B1 was the pioneer high-performance membrane model and B2 was a model that simulated a low-flux cellulosic membrane. The development of B1 led to the development of the dialysis machine with an ultrafiltration rate (UFR)-controlling function because the UFR of B1 was too high to control using transmembrane pressure control. B1 was used not only as a dialyzer but also as a hemodiafilter by combination with a UFR controller. Biocompatibility of the dialysis membrane, complement activation and/or transient leukopenia was studied with B2. Cooperative studies between Niigata University and Toray resulted in Gejyo's finding regarding the harmfulness of β(2)-microglobulin (BMG). Long-term follow-up of patients dialyzed using the BK membrane revealed that plasma BMG levels were significantly low and that the occurrence ratio of carpal tunnel syndrome was suppressed. These results were obtained by the adsorptive removal of BMG onto a PMMA membrane. Several papers have discussed new aspects in succession mainly based on clinical experiences that were not aimed at a development stage, i.e. they were kinds of serendipity. For the BK-F membrane with the largest pore size, this includes anemia and removal or modification of furancarboxylic acid, homocysteine, pentosidine and soluble CD40. For the BG membrane with a slightly anionic component, this includes pruritus and removal of free immunoglobulin light chains. Even patients' prognoses may be modified by the use of PMMA membrane. The mechanisms of these findings have been clarified bit by bit and the membrane will further open new frontiers in dialysis treatment. Copyright © 2011 S. Karger AG, Basel.

High-flux ceramic membranes with a nanomesh of metal oxide nanofibers.

PubMed

Ke, Xue Bin; Zheng, Zhan Feng; Liu, Hong Wei; Zhu, Huai Yong; Gao, Xue Ping; Zhang, Li Xiong; Xu, Nan Ping; Wang, Huanting; Zhao, Hui Jun; Shi, Jeffrey; Ratinac, Kyle R

2008-04-24

Traditional ceramic separation membranes, which are fabricated by applying colloidal suspensions of metal hydroxides to porous supports, tend to suffer from pinholes and cracks that seriously affect their quality. Other intrinsic problems for these membranes include dramatic losses of flux when the pore sizes are reduced to enhance selectivity and dead-end pores that make no contribution to filtration. In this work, we propose a new strategy for addressing these problems by constructing a hierarchically structured separation layer on a porous substrate using large titanate nanofibers and smaller boehmite nanofibers. The nanofibers are able to divide large voids into smaller ones without forming dead-end pores and with the minimum reduction of the total void volume. The separation layer of nanofibers has a porosity of over 70% of its volume, whereas the separation layer in conventional ceramic membranes has a porosity below 36% and inevitably includes dead-end pores that make no contribution to the flux. This radical change in membrane texture greatly enhances membrane performance. The resulting membranes were able to filter out 95.3% of 60-nm particles from a 0.01 wt % latex while maintaining a relatively high flux of between 800 and 1000 L/m2.h, under a low driving pressure (20 kPa). Such flow rates are orders of magnitude greater than those of conventional membranes with equal selectivity. Moreover, the flux was stable at approximately 800 L/m2.h with a selectivity of more than 95%, even after six repeated runs of filtration and calcination. Use of different supports, either porous glass or porous alumina, had no substantial effect on the performance of the membranes; thus, it is possible to construct the membranes from a variety of supports without compromising functionality. The Darcy equation satisfactorily describes the correlation between the filtration flux and the structural parameters of the new membranes. The assembly of nanofiber meshes to combine high flux with excellent selectivity is an exciting new direction in membrane fabrication.

Identification of Residues in the Lipopolysaccharide ABC Transporter That Coordinate ATPase Activity with Extractor Function.

PubMed

Simpson, Brent W; Owens, Tristan W; Orabella, Matthew J; Davis, Rebecca M; May, Janine M; Trauger, Sunia A; Kahne, Daniel; Ruiz, Natividad

2016-10-18

The surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS), creating a permeability barrier against toxic molecules, including many antimicrobials. To assemble LPS on their surface, Gram-negative bacteria must extract newly synthesized LPS from the inner membrane, transport it across the aqueous periplasm, and translocate it across the outer membrane. The LptA to -G proteins assemble into a transenvelope complex that transports LPS from the inner membrane to the cell surface. The Lpt system powers LPS transport from the inner membrane by using a poorly characterized ATP-binding cassette system composed of the ATPase LptB and the transmembrane domains LptFG. Here, we characterize a cluster of residues in the groove region of LptB that is important for controlling LPS transport. We also provide the first functional characterization of LptFG and identify their coupling helices that interact with the LptB groove. Substitutions at conserved residues in these coupling helices compromise both the assembly and function of the LptB 2 FG complex. Defects in LPS transport conferred by alterations in the LptFG coupling helices can be rescued by changing a residue in LptB that is adjacent to functionally important residues in the groove region. This suppression is achieved by increasing the ATPase activity of the LptB 2 FG complex. Taken together, these data identify a specific binding site in LptB for the coupling helices of LptFG that is responsible for coupling of ATP hydrolysis by LptB with LptFG function to achieve LPS extraction. Lipopolysaccharide (LPS) is synthesized at the cytoplasmic membrane of Gram-negative bacteria and transported across several compartments to the cell surface, where it forms a barrier that protects these organisms from antibiotics. The LptB 2 FG proteins form an ATP-binding cassette (ABC) transporter that uses energy from ATP hydrolysis in the cytoplasm to facilitate extraction of LPS from the outer face of the cytoplasmic membrane prior to transport to the cell surface. How ATP hydrolysis is coupled with LPS release from the membrane is not understood. We have identified residues at the interface between the ATPase and the transmembrane domains of this heteromeric ABC complex that are important for LPS transport, some of which coordinate ATPase activity with LPS release. Copyright © 2016 Simpson et al.

Drug Transporters and Na+/H+ Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

PubMed Central

Walsh, Dustin R.; Nolin, Thomas D.

2015-01-01

Drug transporters govern the absorption, distribution, and elimination of pharmacologically active compounds. Members of the solute carrier and ATP binding-cassette drug transporter family mediate cellular drug uptake and efflux processes, thereby coordinating the vectorial movement of drugs across epithelial barriers. To exert their physiologic and pharmacological function in polarized epithelia, drug transporters must be targeted and stabilized to appropriate regions of the cell membrane (i.e., apical versus basolateral). Despite the critical importance of drug transporter membrane targeting, the mechanisms that underlie these processes are largely unknown. Several clinically significant drug transporters possess a recognition sequence that binds to PSD-95/Drosophila discs large/ZO-1 (PDZ) proteins. PDZ proteins, such as the Na+/H+ exchanger regulatory factor (NHERF) family, act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is particularly high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia, tissues important to drug disposition. Several recent studies have highlighted NHERF proteins as determinants of drug transporter function secondary to their role in controlling membrane abundance and localization. Mounting evidence strongly suggests that NHERF proteins may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development. PMID:26092975

Membrane on a Chip: A Functional Tethered Lipid Bilayer Membrane on Silicon Oxide Surfaces

PubMed Central

Atanasov, Vladimir; Knorr, Nikolaus; Duran, Randolph S.; Ingebrandt, Sven; Offenhäusser, Andreas; Knoll, Wolfgang; Köper, Ingo

2005-01-01

Tethered membranes have been proven during recent years to be a powerful and flexible biomimetic platform. We reported in a previous article on the design of a new architecture based on the self-assembly of a thiolipid on ultrasmooth gold substrates, which shows extremely good electrical sealing properties as well as functionality of a bilayer membrane. Here, we describe the synthesis of lipids for a more modular design and the adaptation of the linker part to silane chemistry. We were able to form a functional tethered bilayer lipid membrane with good electrical sealing properties covering a silicon oxide surface. We demonstrate the functional incorporation of the ion carrier valinomycin and of the ion channel gramicidin. PMID:16127170

Surface dynamics of voltage-gated ion channels.

PubMed

Heine, Martin; Ciuraszkiewicz, Anna; Voigt, Andreas; Heck, Jennifer; Bikbaev, Arthur

2016-07-03

Neurons encode information in fast changes of the membrane potential, and thus electrical membrane properties are critically important for the integration and processing of synaptic inputs by a neuron. These electrical properties are largely determined by ion channels embedded in the membrane. The distribution of most ion channels in the membrane is not spatially uniform: they undergo activity-driven changes in the range of minutes to days. Even in the range of milliseconds, the composition and topology of ion channels are not static but engage in highly dynamic processes including stochastic or activity-dependent transient association of the pore-forming and auxiliary subunits, lateral diffusion, as well as clustering of different channels. In this review we briefly discuss the potential impact of mobile sodium, calcium and potassium ion channels and the functional significance of this for individual neurons and neuronal networks.

Anatomical influences on internally coupled ears in reptiles.

PubMed

Young, Bruce A

2016-10-01

Many reptiles, and other vertebrates, have internally coupled ears in which a patent anatomical connection allows pressure waves generated by the displacement of one tympanic membrane to propagate (internally) through the head and, ultimately, influence the displacement of the contralateral tympanic membrane. The pattern of tympanic displacement caused by this internal coupling can give rise to novel sensory cues. The auditory mechanics of reptiles exhibit more anatomical variation than in any other vertebrate group. This variation includes structural features such as diverticula and septa, as well as coverings of the tympanic membrane. Many of these anatomical features would likely influence the functional significance of the internal coupling between the tympanic membranes. Several of the anatomical components of the reptilian internally coupled ear are under active motor control, suggesting that in some reptiles the auditory system may be more dynamic than previously recognized.

Simultaneous AFM topography and recognition imaging at the plasma membrane of mammalian cells.

PubMed

Chtcheglova, Lilia A; Hinterdorfer, Peter

2018-01-01

Elucidation the nano-organization of membrane proteins at/within the plasma membrane is probably the most demanding and still challenging task in cell biology since requires experimental approaches with nanoscale resolution. During last decade, atomic force microscopy (AFM)-based simultaneous topography and recognition imaging (TREC) has become a powerful tool to quickly obtain local receptor nano-maps on complex heterogeneous biosurfaces such as cells and membranes. Here we emphasize the TREC technique and explain how to unravel the nano-landscape of mammalian cells. We describe the procedures for all steps of the experiment including tip functionalization with ligand molecules, sample preparation, and localization of key molecules on the cell surface. We also discuss the current limitations and future perspectives of this technique. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Structural basis of membrane disruption and cellular toxicity by α-synuclein oligomers.

PubMed

Fusco, Giuliana; Chen, Serene W; Williamson, Philip T F; Cascella, Roberta; Perni, Michele; Jarvis, James A; Cecchi, Cristina; Vendruscolo, Michele; Chiti, Fabrizio; Cremades, Nunilo; Ying, Liming; Dobson, Christopher M; De Simone, Alfonso

2017-12-15

Oligomeric species populated during the aggregation process of α-synuclein have been linked to neuronal impairment in Parkinson's disease and related neurodegenerative disorders. By using solution and solid-state nuclear magnetic resonance techniques in conjunction with other structural methods, we identified the fundamental characteristics that enable toxic α-synuclein oligomers to perturb biological membranes and disrupt cellular function; these include a highly lipophilic element that promotes strong membrane interactions and a structured region that inserts into lipid bilayers and disrupts their integrity. In support of these conclusions, mutations that target the region that promotes strong membrane interactions by α-synuclein oligomers suppressed their toxicity in neuroblastoma cells and primary cortical neurons. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Surface dynamics of voltage-gated ion channels

PubMed Central

Heine, Martin; Ciuraszkiewicz, Anna; Voigt, Andreas; Heck, Jennifer; Bikbaev, Arthur

2016-01-01

ABSTRACT Neurons encode information in fast changes of the membrane potential, and thus electrical membrane properties are critically important for the integration and processing of synaptic inputs by a neuron. These electrical properties are largely determined by ion channels embedded in the membrane. The distribution of most ion channels in the membrane is not spatially uniform: they undergo activity-driven changes in the range of minutes to days. Even in the range of milliseconds, the composition and topology of ion channels are not static but engage in highly dynamic processes including stochastic or activity-dependent transient association of the pore-forming and auxiliary subunits, lateral diffusion, as well as clustering of different channels. In this review we briefly discuss the potential impact of mobile sodium, calcium and potassium ion channels and the functional significance of this for individual neurons and neuronal networks. PMID:26891382

Life at the border: Adaptation of proteins to anisotropic membrane environment

PubMed Central

Pogozheva, Irina D; Mosberg, Henry I; Lomize, Andrei L

2014-01-01

This review discusses main features of transmembrane (TM) proteins which distinguish them from water-soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co-translational and post-translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large-scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen-bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side-chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding. PMID:24947665

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

Kusumastuti, Ella, E-mail: ella.kusuma@gmail.com; Siniwi, Widasari Trisna, E-mail: wsiniwi@gmail.com; Mahatmanti, F. Widhi

Chitosan has been widely used as polymer matrix for Polymer Electrolyte Membrane (PEM) application replacing Nafion which has shortcomings in terms of high methanol permeability that degrades the performance of fuel cells. Chitosan membranes modification is performed by adding nanosilica to prevent methanol transport through the membrane. Nanosilica is synthesized by sol-gel method and the particle diameter is obtained by analysis using Breunner Emmet Teller (BET) that is 6.59 nm. Nanosilica is mixed with chitosan solution to obtain nanosilica-chitosan as polymer electrolyte membrane. The membranes are synthesized through phase inversion method with nanosilica composition including 0; 0.5; 1; 2; 3; 5;more » and 10% w/w of chitosan. Characterization of the membranes indicate that the results of water swelling, proton conductivity and methanol permeability of the membrane with 3% nanosilica respectively were 49.23%, 0.231 S/cm, and 5.43 x 10{sup −7} cm{sup 2}/s. Based on the results of membrane selectivity calculation, the optimum membrane is the composition of 3% nanosilica with value 5.91 x 105 S s cm{sup −3}. The results of functional groups analysis with FTIR showed that it was only physical interaction that occurred between chitosan and nanosilica since no significant changes found in peak around the wave number 1000-1250 cm{sup −-1}.« less

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

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed Central

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-01-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria. PMID:10816421

CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

PubMed

Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

2000-06-01

The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

Nanomaterials for membrane fouling control: accomplishments and challenges.

PubMed

Yang, Qian; Mi, Baoxia

2013-11-01

We report a review of recent research efforts on incorporating nanomaterials-including metal/metal oxide nanoparticles, carbon-based nanomaterials, and polymeric nanomaterials-into/onto membranes to improve membrane antifouling properties in biomedical or potentially medical-related applications. In general, nanomaterials can be incorporated into/onto a membrane by blending them into membrane fabricating materials or by attaching them to membrane surfaces via physical or chemical approaches. Overall, the fascinating, multifaceted properties (eg, high hydrophilicity, superparamagnetic properties, antibacterial properties, amenable functionality, strong hydration capability) of nanomaterials provide numerous novel strategies and unprecedented opportunities to fully mitigate membrane fouling. However, there are still challenges in achieving a broader adoption of nanomaterials in the membrane processes used for biomedical applications. Most of these challenges arise from the concerns over their long-term antifouling performance, hemocompatibility, and toxicity toward humans. Therefore, rigorous investigation is still needed before the adoption of some of these nanomaterials in biomedical applications, especially for those nanomaterials proposed to be used in the human body or in contact with living tissue/body fluids for a long period of time. Nevertheless, it is reasonable to predict that the service lifetime of membrane-based biomedical devices and implants will be prolonged significantly with the adoption of appropriate fouling control strategies. Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

Testing of various membranes for use in a novel sediment porewater isolation chamber for infaunal invertebrate exposure to PCBs.

PubMed

Coleman, Jessica G; Lotufo, Guilherme R; Kennedy, Alan J; Poda, Aimee R; Rushing, Todd S; Ruiz, Carlos E; Bridges, Todd S

2014-07-01

In benthic sediment bioassays, determining the relative contribution to exposure by contaminants in overlying water, porewater, and sediment particles is technically challenging. The purpose of the present study was to assess the potential for membranes to be utilized as a mechanism to allow freely dissolved hydrophobic organic contaminants into a pathway isolation exposure chamber (PIC) while excluding all sediment particles and dissolved organic carbon (DOC). This investigation was conducted in support of a larger effort to assess contaminant exposure pathways to benthos. While multiple passive samplers exist for estimating concentrations of contaminants in porewater such as those using solid-phase micro extraction (SPME) and polyoxymethylene (POM), techniques to effectively isolate whole organism exposure to porewater within a sediment system are not available. We tested the use of four membranes of different pore sizes (0.1-1.2μm) including nylon, polycarbonate, polyethylsulfone, and polytetrafluoroethylene with a hydrophilic coating. Exposures included both diffusion of radiolabeled and non-labeled contaminants across membranes from aqueous, sediment slurry, and whole sediment sources to assess and evaluate the best candidate membrane. Data generated from the present study was utilized to select the most suitable membrane for use in the larger bioavailability project which sought to assess the relevance of functional ecology in bioavailability of contaminated sediments at remediation sites. The polytetrafluoroethylene membrane was selected for use in the PIC, although exclusion of dissolved organic carbon was not achieved. Published by Elsevier Ltd.

Genes for all metals--a bacterial view of the periodic table. The 1996 Thom Award Lecture.

PubMed

Silver, S

1998-01-01

Bacterial chromosomes have genes for transport proteins for inorganic nutrient cations and oxyanions, such as NH4+, K+, Mg2+, Co2+, Fe3+, Mn2+, Zn2+ and other trace cations, and PO4(3-), SO4(2-) and less abundant oxyanions. Together these account for perhaps a few hundred genes in many bacteria. Bacterial plasmids encode resistance systems for toxic metal and metalloid ions including Ag+, AsO2-, AsO4(3-), Cd2+, Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. Most resistance systems function by energy-dependent efflux of toxic ions. A few involve enzymatic (mostly redox) transformations. Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. The Cd(2+)-resistance cation pump of Gram-positive bacteria is membrane P-type ATPase, which has been labeled with 32P from [gamma-32P]ATP and drives ATP-dependent Cd2+ (and Zn2+) transport by membrane vesicles. The genes defective in the human hereditary diseases of copper metabolism, Menkes syndrome and Wilson's disease, encode P-type ATPases that are similar to bacterial cadmium ATPases. The arsenic resistance system transports arsenite [As(III)], alternatively with the ArsB polypeptide functioning as a chemiosmotic efflux transporter or with two polypeptides, ArsB and ArsA, functioning as an ATPase. The third protein of the arsenic resistance system is an enzyme that reduces intracellular arsenate [As(V)] to arsenite [As(III)], the substrate of the efflux system. In Gram-negative cells, a three polypeptide complex functions as a chemiosmotic cation/protein exchanger to efflux Cd2+, Zn2+ and Co2+. This pump consists of an inner membrane (CzcA), an outer membrane (CzcC) and a membrane-spanning (CzcB) protein that function together.

Discovery of CLC transport proteins: cloning, structure, function and pathophysiology

PubMed Central

Jentsch, Thomas J

2015-01-01

Abstract After providing a personal description of the convoluted path leading 25 years ago to the molecular identification of the Torpedo Cl− channel ClC-0 and the discovery of the CLC gene family, I succinctly describe the general structural and functional features of these ion transporters before giving a short overview of mammalian CLCs. These can be categorized into plasma membrane Cl− channels and vesicular Cl−/H+-exchangers. They are involved in the regulation of membrane excitability, transepithelial transport, extracellular ion homeostasis, endocytosis and lysosomal function. Diseases caused by CLC dysfunction include myotonia, neurodegeneration, deafness, blindness, leukodystrophy, male infertility, renal salt loss, kidney stones and osteopetrosis, revealing a surprisingly broad spectrum of biological roles for chloride transport that was unsuspected when I set out to clone the first voltage-gated chloride channel. PMID:25590607

Alport syndrome and Pierson syndrome: Diseases of the glomerular basement membrane.

PubMed

Funk, Steven D; Lin, Meei-Hua; Miner, Jeffrey H

2018-04-16

The glomerular basement membrane (GBM) is an important component of the kidney's glomerular filtration barrier. Like all basement membranes, the GBM contains type IV collagen, laminin, nidogen, and heparan sulfate proteoglycan. It is flanked by the podocytes and glomerular endothelial cells that both synthesize it and adhere to it. Mutations that affect the GBM's collagen α3α4α5(IV) components cause Alport syndrome (kidney disease with variable ear and eye defects) and its variants, including thin basement membrane nephropathy. Mutations in LAMB2 that impact the synthesis or function of laminin α5β2γ1 (LM-521) cause Pierson syndrome (congenital nephrotic syndrome with eye and neurological defects) and its less severe variants, including isolated congenital nephrotic syndrome. The very different types of kidney diseases that result from mutations in collagen IV vs. laminin are likely due to very different pathogenic mechanisms. A better understanding of these mechanisms should lead to targeted therapeutic approaches that can help people with these rare but important diseases. Copyright © 2017 International Society of Matrix Biology. Published by Elsevier B.V. All rights reserved.

Targeting the membrane-anchored serine protease testisin with a novel engineered anthrax toxin prodrug to kill tumor cells and reduce tumor burden

PubMed Central

Martin, Erik W.; Buzza, Marguerite S.; Driesbaugh, Kathryn H.; Liu, Shihui; Fortenberry, Yolanda M.; Leppla, Stephen H.; Antalis, Toni M.

2015-01-01

The membrane-anchored serine proteases are a unique group of trypsin-like serine proteases that are tethered to the cell surface via transmembrane domains or glycosyl-phosphatidylinositol-anchors. Overexpressed in tumors, with pro-tumorigenic properties, they are attractive targets for protease-activated prodrug-like anti-tumor therapies. Here, we sought to engineer anthrax toxin protective antigen (PrAg), which is proteolytically activated on the cell surface by the proprotein convertase furin to instead be activated by tumor cell-expressed membrane-anchored serine proteases to function as a tumoricidal agent. PrAg's native activation sequence was mutated to a sequence derived from protein C inhibitor (PCI) that can be cleaved by membrane-anchored serine proteases, to generate the mutant protein PrAg-PCIS. PrAg-PCIS was resistant to furin cleavage in vitro, yet cytotoxic to multiple human tumor cell lines when combined with FP59, a chimeric anthrax toxin lethal factor-Pseudomonas exotoxin fusion protein. Molecular analyses showed that PrAg-PCIS can be cleaved in vitro by several serine proteases including the membrane-anchored serine protease testisin, and mediates increased killing of testisin-expressing tumor cells. Treatment with PrAg-PCIS also potently attenuated the growth of testisin-expressing xenograft tumors in mice. The data indicates PrAg can be engineered to target tumor cell-expressed membrane-anchored serine proteases to function as a potent tumoricidal agent. PMID:26392335

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

PubMed

Putta, Priya; Rankenberg, Johanna; Korver, Ruud A; van Wijk, Ringo; Munnik, Teun; Testerink, Christa; Kooijman, Edgar E

2016-11-01

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins. Copyright © 2016. Published by Elsevier B.V.

Cathepsin S Contributes to the Pathogenesis of Muscular Dystrophy in Mice.

PubMed

Tjondrokoesoemo, Andoria; Schips, Tobias G; Sargent, Michelle A; Vanhoutte, Davy; Kanisicak, Onur; Prasad, Vikram; Lin, Suh-Chin J; Maillet, Marjorie; Molkentin, Jeffery D

2016-05-06

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin protein compromises the stability of the sarcolemma membrane surrounding each muscle cell fiber, leading to membrane ruptures and leakiness that induces myofiber necrosis, a subsequent inflammatory response, and progressive tissue fibrosis with loss of functional capacity. Cathepsin S (Ctss) is a cysteine protease that is actively secreted in areas of tissue injury and ongoing inflammation, where it participates in extracellular matrix remodeling and healing. Here we show significant induction of Ctss expression and proteolytic activity following acute muscle injury or in muscle from mdx mice, a model of DMD. To examine the functional ramifications associated with greater Ctss expression, the Ctss gene was deleted in the mdx genetic background, resulting in protection from muscular dystrophy pathogenesis that included reduced myofiber turnover and histopathology, reduced fibrosis, and improved running capacity. Mechanistically, deletion of the Ctss gene in the mdx background significantly increased myofiber sarcolemmal membrane stability with greater expression and membrane localization of utrophin, integrins, and β-dystroglycan, which anchor the membrane to the basal lamina and underlying cytoskeletal proteins. Consistent with these results, skeletal muscle-specific transgenic mice overexpressing Ctss showed increased myofiber necrosis, muscle histopathology, and a functional deficit reminiscent of muscular dystrophy. Hence, Ctss induction during muscular dystrophy is a pathologic event that partially underlies disease pathogenesis, and its inhibition might serve as a new therapeutic strategy in DMD. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Domain-swapping analysis of FtsI, FtsL, and FtsQ, bitopic membrane proteins essential for cell division in Escherichia coli.

PubMed Central

Guzman, L M; Weiss, D S; Beckwith, J

1997-01-01

FtsI, FtsL, and FtsQ are three membrane proteins required for assembly of the division septum in the bacterium Escherichia coli. Cells lacking any of these three proteins form long, aseptate filaments that eventually lyse. FtsI, FtsL, and FtsQ are not homologous but have similar overall structures: a small cytoplasmic domain, a single membrane-spanning segment (MSS), and a large periplasmic domain that probably encodes the primary functional activities of these proteins. The periplasmic domain of FtsI catalyzes transpeptidation and is involved in the synthesis of septal peptidoglycan. The precise functions of FtsL and FtsQ are not known. To ask whether the cytoplasmic domain and MSS of each protein serve only as a membrane anchor or have instead a more sophisticated function, we have used molecular genetic techniques to swap these domains among the three Fts proteins and one membrane protein not involved in cell division, MalF. In the cases of FtsI and FtsL, replacement of the cytoplasmic domain and/or MSS resulted in the loss of the ability to support cell division. For FtsQ, MSS swaps supported cell division but cytoplasmic domain swaps did not. We discuss several potential interpretations of these results, including that the essential domains of FtsI, FtsL, and FtsQ have a role in regulating the localization and/or activity of these proteins to ensure that septum formation occurs at the right place in the cell and at the right time during the division cycle. PMID:9260951

Submerged anaerobic membrane bioreactor for wastewater treatment and energy generation.

PubMed

Bornare, J B; Adhyapak, U S; Minde, G P; Kalyan Raman, V; Sapkal, V S; Sapkal, R S

2015-01-01

Compared with conventional wastewater treatment processes, membrane bioreactors (MBRs) offer several advantages including high biodegradation efficiency, excellent effluent quality and smaller footprint. However, it has some limitations on account of its energy intensive operation. In recent years, there has been growing interest in use of anaerobic membrane bioreactors (AnMBRs) due to their potential advantages over aerobic systems, which include low sludge production and energy generation in terms of biogas. The aim of this study was to evaluate the performance of a submerged AnMBR for the treatment of synthetic wastewater having 4,759 mg/l chemical oxygen demand (COD). The COD removal efficiency was over 95% during the performance evaluation study. Treated effluent with COD concentration of 231 mg/l was obtained for 25.5 hours hydraulic retention time. The obtained total organic carbon concentrations in feed and permeate were 1,812 mg/l and 89 mg/l, respectively. An average biogas generation and yield were 25.77 l/d and 0.36 m3/kg COD, respectively. Evolution of trans-membrane pressure (TMP) as a function of time was studied and an average TMP of 15 kPa was found suitable to achieve membrane flux of 12.17 l/(m2h). Almost weekly back-flow chemical cleaning of the membrane was found necessary to control TMP within the permissible limit of 20 kPa.

Sheet Membrane Spacesuit Water Membrane Evaporator Thermal Test

NASA Technical Reports Server (NTRS)

Trevino, Luis A.; Bue, Grant C.

2009-01-01

For future lunar extravehicular activities (EVA), one method under consideration for rejecting crew and electronics heat involves evaporating water through a hydrophobic, porous Teflon(Registered Trademark) membrane. A Spacesuit Water Membrane Evaporator (SWME) prototype using this membrane was successfully tested by Ungar and Thomas (2001) with predicted performance matching test data well. The above referenced work laid the foundation for the design of a compact sheet membrane SWME development unit for use in the Constellation System Spacesuit Element Portable Life Support System (Vogel and et. al., ICES 2008). Major design objectives included minimizing mass, volume, and manufacturing complexity while rejecting a minimum of 810 watts of heat from water flowing through the SWME at 91 kg/hr with an inlet temperature of 291K. The design meeting these objectives consisted of three concentric cylindrical water channels interlaced with four water vapor channels. Two units were manufactured for the purpose of investigating manufacturing techniques and performing thermal testing. The extensive thermal test measured SWME heat rejection as a function of water inlet temperatures, water flow-rates, water absolute pressures, water impurities, and water vapor back-pressures. This paper presents the test results and subsequent analysis, which includes a comparison of SWME heat rejection measurements to pretest predictions. In addition, test measurements were taken such that an analysis of the commercial-off-the-shelf vapor pressure control valve could be performed.

Phytocannabinoids and endocannabinoids.

PubMed

Fisar, Zdenek

2009-01-01

Progress in understanding the molecular mechanisms of cannabis action was made after discovery of cannabinoid receptors in the brain and the finding of endogenous metabolites with affinity to them. Activation of cannabinoid receptors on synaptic terminals results in regulation of ion channels, neurotransmitter release and synaptic plasticity. Neuromodulation of synapses by the cannabinoids is proving to have a wide range of functional effects, making them potential targets as medical preparations in a variety of illnesses, including some mental disorders and neurodegenerative illnesses. Cannabis contains a large amount of substances with affinity for the cannabinoid receptors. The endocannabinoids are a family of lipid neurotransmitters that engage the same membrane receptors targeted by tetrahydrocannabinol and that mediate retrograde signal from postsynaptic neurons to presynaptic ones. Discovery of endogenous cannabinoids and studies of the physiological functions of the cannabinoid system in the brain and body are producing a number of important findings about the role of membrane lipids and fatty acids in nerve signal transduction. Plant, endogenous and synthetic cannabinoids are using in these studies. The role of lipid membranes in the cannabinoid system follows from the fact that the source and supply of endogenous cannabinoids are derived from arachidonic acid, an important membrane constituent. The study of structure-activity relationships of molecules which influence the cannabinoid system in the brain and body is crucial in search of medical preparations with the therapeutic effects of the phytocannabinoids without the negative effects on cognitive function attributed to cannabis.

Tunable Nanocomposite Membranes for Water Remediation and Separations

NASA Astrophysics Data System (ADS)

Sierra, Sebastian Hernandez

Nano-structured material fabrication using functionalized membranes with polyelectrolytes is a promising research field for water pollution, catalytic and mining applications. These responsive polymers react to external stimuli like temperature, pH, radiation, ionic strength or chemical composition. Such nanomaterials provide novel hybrid properties and can also be self-supported in addition to the membranes. Polyelectrolytes (as hydrogels) have pH responsiveness. The hydrogel moieties gain or lose protons based on the pH, displaying swelling properties. These responsive materials can be exploited to synthesize metal nanoparticles in situ using their functional groups, or to immobilize other polyelectrolytes and biomolecules. Due to their properties, these responsive materials prevent the loss of nanomaterials to the environment and improve reactivity due to their larger surface areas, expanding their range of applications. The present work describes different techniques used to create nanocomposites based on poly(vinylidene fluoride) (PVDF) hollow fiber and flat sheet membranes, both thick sponge-like and thin. Due to their hydrophobicity, hollow fiber membranes were hydrophilized by a water-based green process of cross-linking polyvinylpyrrolidone (PVP) onto their surface. Commercial hydrophilic and hydrophilized lab-prepared membranes were subsequently functionalized with a poly(acrylic acid) (PAA) hydrogel through free radical polymerizations. This work advanced membrane functionalization, specifically flat sheet membranes, from lab-scale to full-scale by modifications of the polymerization procedures. The hydrogel functionalized membranes by redox polymerization showed an expected responsive behavior, represented by permeability variation at various pH values (4.0 ≤ pH ≤ 9.0), from 53.9 to 3.4 L/(m2EhEbar) and a change in effective pore size from 222 to 111 nm, being 3800 L/(m 2EhEbar) and 650 nm the former permeability and pore size values of the non-functionalized membrane. Then, throughout a double ion exchange of sodium/iron and a subsequent reduction, bimetallic Fe/Pd nanoparticles were synthesized in-situ. Similarly, it was possible to use the reacted accelerants of the redox polymerization to synthesize Fe0 nanoparticles. These hydrogel-membrane systems with Fe/Pd nanoparticles were studied throughout the reduction of trichloroethylene (TCE). This work has demonstrated an effective improvement in TCE reduction by the variation of the supporting membrane types and the functionalization (polymerization and nanoparticle synthesis) processes. The TCE normalized dechlorination rates (k sa) are 3 times greater and 8 times for hollow fiber and sponge-like flat sheet membranes, respectively, than previous studies. For membrane supported Fe/Pd nanoparticles by redox functionalization, the dechlorination rates are similar to previous works in flat sheet membranes; and for the redox polymerized hydrogel, the dechlorination rates are the highest results with 1.3 times greater than the rates of solution-phase nanoparticles and 10 times the rate values of the membranes. All supports showed nonsignificant nanoparticle loss (up to 1%). Up to 80% of reduction was achieved within 2 hours with chloride production near to stoichiometric values (3:1), demonstrating absence of intermediates. As an extension of the membrane functionalization, it was possible to immobilize Outer membrane protein F precursor (OmpF) from Escherichia coli within the PVDF membrane pore structure, using layer-by-layer (LbL) assembly of polyeletrolytes. This LbL technique allows to reuse the membranes numerous times, having reproducibility and greater selective rejections of uncharged (organic species) over charged solutes (small ions) than similar functionalized membranes without OmpF: 1.7 times and 2.0 times higher for Organic/CaCl2 and Organic/NaCl, respectively. Additionally, the permeability of OmpFmembranes is almost double of the non-OmpF: 2.6 to 1.5 L/(m2˙h˙bar).

Time-Resolved Neutron Interferometry and the Mechanism of Electromechanical Coupling in Voltage-Gated Ion Channels.

PubMed

Blasie, J Kent

2018-01-01

The mechanism of electromechanical coupling for voltage-gated ion channels (VGICs) involved in neurological signal transmission, primarily Nav- and Kv-channels, remains unresolved. Anesthetics have been shown to directly impact this mechanism, at least for Kv-channels. Molecular dynamics computer simulations can now predict the structures of VGICs embedded within a hydrated phospholipid bilayer membrane as a function of the applied transmembrane voltage, but significant assumptions are still necessary. Nevertheless, these simulations are providing new insights into the mechanism of electromechanical coupling at the atomic level in 3-D. We show that time-resolved neutron interferometry can be used to investigate directly the profile structure of a VGIC, vectorially oriented within a single hydrated phospholipid bilayer membrane at the solid-liquid interface, as a function of the applied transmembrane voltage in the absence of any assumptions or potentially perturbing modifications of the VGIC protein and/or the host membrane. The profile structure is a projection of the membrane's 3-D structure onto the membrane normal and, in the absence of site-directed deuterium labeling, is provided at substantially lower spatial resolution than the atomic level. Nevertheless, this novel approach can be used to directly test the validity of the predictions from molecular dynamics simulations. We describe the key elements of our novel experimental approach, including why each is necessary and important to providing the essential information required for this critical comparison of "simulation" vs "experiment." In principle, the approach could be extended to higher spatial resolution and to include the effects of anesthetics on the electromechanical coupling mechanism in VGICs. © 2018 Elsevier Inc. All rights reserved.

Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae

PubMed Central

Henry, Susan A.; Kohlwein, Sepp D.; Carman, George M.

2012-01-01

Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces cerevisiae is a model system of choice for the study of the genetics, biochemistry, and cell biology of eukaryotic lipid metabolism. Glycerolipids (e.g., phospholipids and triacylglycerol) and their precursors are synthesized and metabolized by enzymes associated with the cytosol and membranous organelles, including endoplasmic reticulum, mitochondria, and lipid droplets. Genetic and biochemical analyses have revealed that glycerolipids play important roles in cell signaling, membrane trafficking, and anchoring of membrane proteins in addition to membrane structure. The expression of glycerolipid enzymes is controlled by a variety of conditions including growth stage and nutrient availability. Much of this regulation occurs at the transcriptional level and involves the Ino2–Ino4 activation complex and the Opi1 repressor, which interacts with Ino2 to attenuate transcriptional activation of UASINO-containing glycerolipid biosynthetic genes. Cellular levels of phosphatidic acid, precursor to all membrane phospholipids and the storage lipid triacylglycerol, regulates transcription of UASINO-containing genes by tethering Opi1 to the nuclear/endoplasmic reticulum membrane and controlling its translocation into the nucleus, a mechanism largely controlled by inositol availability. The transcriptional activator Zap1 controls the expression of some phospholipid synthesis genes in response to zinc availability. Regulatory mechanisms also include control of catalytic activity of glycerolipid enzymes by water-soluble precursors, products and lipids, and covalent modification of phosphorylation, while in vivo function of some enzymes is governed by their subcellular location. Genome-wide genetic analysis indicates coordinate regulation between glycerolipid metabolism and a broad spectrum of metabolic pathways. PMID:22345606

Functions of a new photoreceptor membrane. [energy conversion via halobacteria rhodopsin changes

NASA Technical Reports Server (NTRS)

Oesterhelt, D.; Stoeckenius, W.

1973-01-01

In the investigation of light responses on halobacteria phototaxis; ATP synthesis; and changes in O2 consumption, purple membrane biosynthesis, and proton translocation were found. The last three effects are discussed, which suggest that the purple membrane may function as an energy-coupling membrane for light. It is also suggested that purple membrane, through cyclic light-induced conformational changes of its bacteriorhodopsin, directly converts absorbed light energy into a proton gradient and presumably also an electric potential difference across the membrane analogous to observations in other prokaryotic cells, mitochondria, and chloroplasts.

Multifunctional nanocomposite hollow fiber membranes by solvent transfer induced phase separation.

PubMed

Haase, Martin F; Jeon, Harim; Hough, Noah; Kim, Jong Hak; Stebe, Kathleen J; Lee, Daeyeon

2017-11-01

The decoration of porous membranes with a dense layer of nanoparticles imparts useful functionality and can enhance membrane separation and anti-fouling properties. However, manufacturing of nanoparticle-coated membranes requires multiple steps and tedious processing. Here, we introduce a facile single-step method in which bicontinuous interfacially jammed emulsions are used to form nanoparticle-functionalized hollow fiber membranes. The resulting nanocomposite membranes prepared via solvent transfer-induced phase separation and photopolymerization have exceptionally high nanoparticle loadings (up to 50 wt% silica nanoparticles) and feature densely packed nanoparticles uniformly distributed over the entire membrane surfaces. These structurally well-defined, asymmetric membranes facilitate control over membrane flux and selectivity, enable the formation of stimuli responsive hydrogel nanocomposite membranes, and can be easily modified to introduce antifouling features. This approach forms a foundation for the formation of advanced nanocomposite membranes comprising diverse building blocks with potential applications in water treatment, industrial separations and as catalytic membrane reactors.

Anatomic and physiologic changes of the aging kidney.

PubMed

Karam, Zeina; Tuazon, Jennifer

2013-08-01

Aging is associated with structural and functional changes in the kidney. Structural changes include glomerulosclerosis, thickening of the basement membrane, increase in mesangial matrix, tubulointerstitial fibrosis and arteriosclerosis. Glomerular filtration rate is maintained until the fourth decade of life, after which it declines. Parallel reductions in renal blood flow occur with redistribution of blood flow from the cortex to the medulla. Other functional changes include an increase in glomerular basement permeability and decreased ability to dilute or concentrate urine. Copyright © 2013 Elsevier Inc. All rights reserved.

[Membrane and functional effects of vinpocetine and tocopherol in rats with experimental cerebral ischemia].

PubMed

Vishnevskiĭ, A A; Korotkevich, I G; Zhaparalieva, Ch O

2009-01-01

The membrane, antioxidant and functional effects of vinpocetine and a-tocopherol have been investigated under conditions of acute experimental cerebral ischemia in rats. Vinpocetine administration decreased accumulation of lysophospholipids in brain plasma membranes. Vinpocetine also blocked accumulation of conjugated dienes (CD). alpha-Tocopherol inhibited augmentation in CD content and did not reduce the level of lysophospholipids in brain plasma membranes. Functional consequences of membrane impairments were also detected in some behavioral tests and physical capabilities. Administration of both vinpocetine and alpha-tocopherol decreased manifestations of the altered parameters induced by cerebral ischemia and vinpocetine was more effective than alpha-tocopherol.

Membrane-type matrix metalloproteases as diverse effectors of cancer progression.

PubMed

Turunen, S Pauliina; Tatti-Bugaeva, Olga; Lehti, Kaisa

2017-11-01

Membrane-type matrix metalloproteases (MT-MMP) are pivotal regulators of cell invasion, growth and survival. Tethered to the cell membranes by a transmembrane domain or GPI-anchor, the six MT-MMPs can exert these functions via cell surface-associated extracellular matrix degradation or proteolytic protein processing, including shedding or release of signaling receptors, adhesion molecules, growth factors and other pericellular proteins. By interactions with signaling scaffold or cytoskeleton, the C-terminal cytoplasmic tail of the transmembrane MT-MMPs further extends their functionality to signaling or structural relay. MT-MMPs are differentially expressed in cancer. The most extensively studied MMP14/MT1-MMP is induced in various cancers along malignant transformation via pathways activated by mutations in tumor suppressors or proto-oncogenes and changes in tumor microenvironment including cellular heterogeneity, extracellular matrix composition, tissue oxygenation, and inflammation. Classically such induction involves transcriptional programs related to epithelial-to-mesenchymal transition. Besides inhibition by endogenous tissue inhibitors, MT-MMP activities are spatially and timely regulated at multiple levels by microtubular vesicular trafficking, dimerization/oligomerization, other interactions and localization in the actin-based invadosomes, in both tumor and the stroma. The functions of MT-MMPs are multifaceted within reciprocal cellular responses in the evolving tumor microenvironment, which poses the importance of these proteases beyond the central function as matrix scissors, and necessitates us to rethink MT-MMPs as dynamic signaling proteases of cancer. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.

WRF-TMH: predicting transmembrane helix by fusing composition index and physicochemical properties of amino acids.

PubMed

Hayat, Maqsood; Khan, Asifullah

2013-05-01

Membrane protein is the prime constituent of a cell, which performs a role of mediator between intra and extracellular processes. The prediction of transmembrane (TM) helix and its topology provides essential information regarding the function and structure of membrane proteins. However, prediction of TM helix and its topology is a challenging issue in bioinformatics and computational biology due to experimental complexities and lack of its established structures. Therefore, the location and orientation of TM helix segments are predicted from topogenic sequences. In this regard, we propose WRF-TMH model for effectively predicting TM helix segments. In this model, information is extracted from membrane protein sequences using compositional index and physicochemical properties. The redundant and irrelevant features are eliminated through singular value decomposition. The selected features provided by these feature extraction strategies are then fused to develop a hybrid model. Weighted random forest is adopted as a classification approach. We have used two benchmark datasets including low and high-resolution datasets. tenfold cross validation is employed to assess the performance of WRF-TMH model at different levels including per protein, per segment, and per residue. The success rates of WRF-TMH model are quite promising and are the best reported so far on the same datasets. It is observed that WRF-TMH model might play a substantial role, and will provide essential information for further structural and functional studies on membrane proteins. The accompanied web predictor is accessible at http://111.68.99.218/WRF-TMH/ .

Thioredoxin h regulates calcium dependent protein kinases in plasma membranes.

PubMed

Ueoka-Nakanishi, Hanayo; Sazuka, Takashi; Nakanishi, Yoichi; Maeshima, Masayoshi; Mori, Hitoshi; Hisabori, Toru

2013-07-01

Thioredoxin (Trx) is a key player in redox homeostasis in various cells, modulating the functions of target proteins by catalyzing a thiol-disulfide exchange reaction. Target proteins of cytosolic Trx-h of higher plants were studied, particularly in the plasma membrane, because plant plasma membranes include various functionally important protein molecules such as transporters and signal receptors. Plasma membrane proteins from Arabidopsis thaliana cell cultures were screened using a resin Trx-h1 mutant-immobilized, and a total of 48 candidate proteins obtained. These included two calcium-sensing proteins: a phosphoinositide-specific phospholipase 2 (AtPLC2) and a calcium-dependent protein kinase 21 (AtCPK21). A redox-dependent change in AtCPK21 kinase activity was demonstrated in vitro. Oxidation of AtCPK21 resulted in a decrease in kinase activity to 19% of that of untreated AtCPK21, but Trx-h1 effectively restored the activity to 90%. An intramolecular disulfide bond (Cys97-Cys108) that is responsible for this redox modulation was then identified. In addition, endogenous AtCPK21 was shown to be oxidized in vivo when the culture cells were treated with H2 O2 . These results suggest that redox regulation of AtCPK21 by Trx-h in response to external stimuli is important for appropriate cellular responses. The relationship between the redox regulation system and Ca(2+) signaling pathways is discussed. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.

Cell surface dynamics - how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton.

PubMed

de Curtis, Ivan; Meldolesi, Jacopo

2012-10-01

Small GTPases are known to regulate hundreds of cell functions. In particular, Rho family GTPases are master regulators of the cytoskeleton. By regulating actin nucleation complexes, Rho GTPases control changes in cell shape, including the extension and/or retraction of surface protrusions and invaginations. Protrusion and invagination of the plasma membrane also involves the interaction between the plasma membrane and the cortical cytoskeleton. This interplay between membranes and the cytoskeleton can lead to an increase or decrease in the plasma membrane surface area and its tension as a result of the fusion (exocytosis) or internalization (endocytosis) of membranous compartments, respectively. For a long time, the cytoskeleton and plasma membrane dynamics were investigated separately. However, studies from many laboratories have now revealed that Rho GTPases, their modulation of the cytoskeleton, and membrane traffic are closely connected during the dynamic remodeling of the cell surface. Arf- and Rab-dependent exocytosis of specific vesicles contributes to the targeting of Rho GTPases and their regulatory factors to discrete sites of the plasma membrane. Rho GTPases regulate the tethering of exocytic vesicles and modulate their subsequent fusion. They also have crucial roles in the different forms of endocytosis, where they participate in the sorting of membrane domains as well as the sculpting and sealing of membrane flasks and cups. Here, we discuss how cell surface dynamics depend on the orchestration of the cytoskeleton and the plasma membrane by Rho GTPases.

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

Lipid Cell Biology: A Focus on Lipids in Cell Division.

PubMed

Storck, Elisabeth M; Özbalci, Cagakan; Eggert, Ulrike S

2018-06-20

Cells depend on hugely diverse lipidomes for many functions. The actions and structural integrity of the plasma membrane and most organelles also critically depend on membranes and their lipid components. Despite the biological importance of lipids, our understanding of lipid engagement, especially the roles of lipid hydrophobic alkyl side chains, in key cellular processes is still developing. Emerging research has begun to dissect the importance of lipids in intricate events such as cell division. This review discusses how these structurally diverse biomolecules are spatially and temporally regulated during cell division, with a focus on cytokinesis. We analyze how lipids facilitate changes in cellular morphology during division and how they participate in key signaling events. We identify which cytokinesis proteins are associated with membranes, suggesting lipid interactions. More broadly, we highlight key unaddressed questions in lipid cell biology and techniques, including mass spectrometry, advanced imaging, and chemical biology, which will help us gain insights into the functional roles of lipids.

Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

PubMed

Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

2012-01-01

Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.

AHNAK1 and AHNAK2 are costameric proteins: AHNAK1 affects transverse skeletal muscle fiber stiffness

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

Marg, Andreas, E-mail: andreas.marg@mdc-berlin.de; Haase, Hannelore; Neumann, Tanja

2010-10-08

Research highlights: {yields} AHNAK1 and AHNAK2 are costameric proteins. {yields} Intact membrane repair in AHNAK1-deficient mice. {yields} AHNAK1{sup -/-} single fibers have a higher transverse stiffness. -- Abstract: The AHNAK scaffold PDZ-protein family is implicated in various cellular processes including membrane repair; however, AHNAK function and subcellular localization in skeletal muscle are unclear. We used specific AHNAK1 and AHNAK2 antibodies to analyzed the detailed localization of both proteins in mouse skeletal muscle. Co-localization of AHNAK1 and AHNAK2 with vinculin clearly demonstrates that both proteins are components of the costameric network. In contrast, no AHNAK expression was detected in the T-tubulemore » system. A laser wounding assay with AHNAK1-deficient fibers suggests that AHNAK1 is not involved in membrane repair. Using atomic force microscopy (AFM), we observed a significantly higher transverse stiffness of AHNAK1{sup -/-} fibers. These findings suggest novel functions of AHNAK proteins in skeletal muscle.« less

Ion transport in pigmentation.

PubMed

Bellono, Nicholas W; Oancea, Elena V

2014-12-01

Skin melanocytes and ocular pigment cells contain specialized organelles called melanosomes, which are responsible for the synthesis of melanin, the major pigment in mammals. Defects in the complex mechanisms involved in melanin synthesis and regulation result in vision and pigmentation deficits, impaired development of the visual system, and increased susceptibility to skin and eye cancers. Ion transport across cellular membranes is critical for many biological processes, including pigmentation, but the molecular mechanisms by which it regulates melanin synthesis, storage, and transfer are not understood. In this review we first discuss ion channels and transporters that function at the plasma membrane of melanocytes; in the second part we consider ion transport across the membrane of intracellular organelles, with emphasis on melanosomes. We discuss recently characterized lysosomal and endosomal ion channels and transporters associated with pigmentation phenotypes. We then review the evidence for melanosomal channels and transporters critical for pigmentation, discussing potential molecular mechanisms mediating their function. The studies investigating ion transport in pigmentation physiology open new avenues for future research and could reveal novel molecular mechanisms underlying melanogenesis.

Ion transport in pigmentation

PubMed Central

Bellono, Nicholas W.; Oancea, Elena V.

2014-01-01

Skin melanocytes and ocular pigment cells contain specialized organelles called melanosomes, which are responsible for the synthesis of melanin, the major pigment in mammals. Defects in the complex mechanisms involved in melanin synthesis and regulation result in vision and pigmentation deficits, impaired development of the visual system,, and increased susceptibility to skin and eye cancers. Ion transport across cellular membranes is critical for many biological processes, including pigmentation, but the molecular mechanisms by which it regulates melanin synthesis, storage, and transfer are not understood. In this review we first discuss ion channels and transporters that function at the plasma membrane of melanocytes; in the second part we consider ion transport across the membrane of intracellular organelles, with emphasis on melanosomes. We discuss recently characterized lysosomal and endosomal ion channels and transporters associated with pigmentation phenotypes. We then review the evidence for melanosomal channels and transporters critical for pigmentation, discussing potential molecular mechanisms mediating their function. The studies investigating ion transport in pigmentation physiology open new avenues for future research and could reveal novel molecular mechanisms underlying melanogenesis. PMID:25034214

Simple, benign, aqueous-based amination of polycarbonate surfaces

DOE PAGES

VanDelinder, Virginia; Wheeler, David R.; Small, Leo J.; ...

2015-03-18

Here we report a simple, safe, environmentally-friendly aqueous method that uses diamines to functionalize a polycarbonate surface with amino groups. We demonstrate the ability of this facile method to serve as a foundation upon which other functionalities may be attached, including anti-fouling coatings and oriented membrane proteins. The use of water as the solvent for the functionalization ensures that solvent induced swelling does not affect the optical or mechanical properties of the polycarbonate.

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.

Cell membrane causes the lipid bilayers to behave as variable capacitors: A resonance with self-induction of helical proteins.

PubMed

Monajjemi, Majid

2015-12-01

Cell membrane has a unique feature of storing biological energies in a physiologically relevant environment. This study illustrates a capacitor model of biological cell membrane including DPPC structures. The electron density profile models, electron localization function (ELF) and local information entropy have been applied to study the interaction of proteins with lipid bilayers in the cell membrane. The quantum and coulomb blockade effects of different thicknesses in the membrane have also been specifically investigated. It has been exhibited the quantum effects can appear in a small region of the free space within the membrane thickness due to the number and type of phospholipid layers. In addition, from the viewpoint of quantum effects by Heisenberg rule, it is shown the quantum tunneling is allowed in some micro positions while it is forbidden in other forms of membrane capacitor systems. Due to the dynamical behavior of the cell membrane, its capacitance is not fixed which results a variable capacitor. In presence of the external fields through protein trance membrane or ions, charges exert forces that can influence the state of the cell membrane. This causes to appear the charge capacitive susceptibility that can resonate with self-induction of helical coils; the resonance of which is the main reason for various biological pulses. Copyright © 2015 Elsevier B.V. All rights reserved.

Lipid Raft Redox Signaling: Molecular Mechanisms in Health and Disease

PubMed Central

Zhou, Fan; Katirai, Foad

2011-01-01

Abstract Lipid rafts, the sphingolipid and cholesterol-enriched membrane microdomains, are able to form different membrane macrodomains or platforms upon stimulations, including redox signaling platforms, which serve as a critical signaling mechanism to mediate or regulate cellular activities or functions. In particular, this raft platform formation provides an important driving force for the assembling of NADPH oxidase subunits and the recruitment of other related receptors, effectors, and regulatory components, resulting, in turn, in the activation of NADPH oxidase and downstream redox regulation of cell functions. This comprehensive review attempts to summarize all basic and advanced information about the formation, regulation, and functions of lipid raft redox signaling platforms as well as their physiological and pathophysiological relevance. Several molecular mechanisms involving the formation of lipid raft redox signaling platforms and the related therapeutic strategies targeting them are discussed. It is hoped that all information and thoughts included in this review could provide more comprehensive insights into the understanding of lipid raft redox signaling, in particular, of their molecular mechanisms, spatial-temporal regulations, and physiological, pathophysiological relevances to human health and diseases. Antioxid. Redox Signal. 15, 1043–1083. PMID:21294649

Proteome-wide identification of predominant subcellular protein localizations in a bacterial model organism.

PubMed

Stekhoven, Daniel J; Omasits, Ulrich; Quebatte, Maxime; Dehio, Christoph; Ahrens, Christian H

2014-03-17

Proteomics data provide unique insights into biological systems, including the predominant subcellular localization (SCL) of proteins, which can reveal important clues about their functions. Here we analyzed data of a complete prokaryotic proteome expressed under two conditions mimicking interaction of the emerging pathogen Bartonella henselae with its mammalian host. Normalized spectral count data from cytoplasmic, total membrane, inner and outer membrane fractions allowed us to identify the predominant SCL for 82% of the identified proteins. The spectral count proportion of total membrane versus cytoplasmic fractions indicated the propensity of cytoplasmic proteins to co-fractionate with the inner membrane, and enabled us to distinguish cytoplasmic, peripheral inner membrane and bona fide inner membrane proteins. Principal component analysis and k-nearest neighbor classification training on selected marker proteins or predominantly localized proteins, allowed us to determine an extensive catalog of at least 74 expressed outer membrane proteins, and to extend the SCL assignment to 94% of the identified proteins, including 18% where in silico methods gave no prediction. Suitable experimental proteomics data combined with straightforward computational approaches can thus identify the predominant SCL on a proteome-wide scale. Finally, we present a conceptual approach to identify proteins potentially changing their SCL in a condition-dependent fashion. The work presented here describes the first prokaryotic proteome-wide subcellular localization (SCL) dataset for the emerging pathogen B. henselae (Bhen). The study indicates that suitable subcellular fractionation experiments combined with straight-forward computational analysis approaches assessing the proportion of spectral counts observed in different subcellular fractions are powerful for determining the predominant SCL of a large percentage of the experimentally observed proteins. This includes numerous cases where in silico prediction methods do not provide any prediction. Avoiding a treatment with harsh conditions, cytoplasmic proteins tend to co-fractionate with proteins of the inner membrane fraction, indicative of close functional interactions. The spectral count proportion (SCP) of total membrane versus cytoplasmic fractions allowed us to obtain a good indication about the relative proximity of individual protein complex members to the inner membrane. Using principal component analysis and k-nearest neighbor approaches, we were able to extend the percentage of proteins with a predominant experimental localization to over 90% of all expressed proteins and identified a set of at least 74 outer membrane (OM) proteins. In general, OM proteins represent a rich source of candidates for the development of urgently needed new therapeutics in combat of resurgence of infectious disease and multi-drug resistant bacteria. Finally, by comparing the data from two infection biology relevant conditions, we conceptually explore methods to identify and visualize potential candidates that may partially change their SCL in these different conditions. The data are made available to researchers as a SCL compendium for Bhen and as an assistance in further improving in silico SCL prediction algorithms. Copyright © 2014 Elsevier B.V. All rights reserved.

Cellulose acetate membranes functionalized with resveratrol by covalent immobilization for improved osseointegration

NASA Astrophysics Data System (ADS)

Pandele, A. M.; Neacsu, P.; Cimpean, A.; Staras, A. I.; Miculescu, F.; Iordache, A.; Voicu, S. I.; Thakur, V. K.; Toader, O. D.

2018-04-01

Covalent immobilization of resveratrol onto cellulose acetate polymeric membranes used as coating on a Mg-1Ca-0.2Mn-0.6Zr alloy is presented for potential application in the improvement of osseointegration processes. For this purpose, cellulose acetate membrane is hydrolysed in the presence of potassium hydroxide, followed by covalent immobilization of aminopropyl triethoxy silane. Resveratrol was immobilized onto membranes using glutaraldehyde as linker. The newly synthesised functional membranes were thoroughly characterized for their structural characteristics determination employing X-ray photoelectron spectroscopy (XPS), infrared spectroscopy (FT-IR), Raman spectroscopy, thermogravimetric analysis (TGA/DTG) and scanning electron microscopy (SEM) techniques. Subsequently, in vitro cellular tests were performed for evaluating the cytotoxicity biocompatibility of synthesized materials and also the osseointegration potential of obtained derivatised membrane material. It was demonstrated that both polymeric membranes support viability and proliferation of the pre-osteoblastic MC3T3-E1 cells, thus providing a good protection against the potential harmful effects of the compounds released from coated alloys. Furthermore, cellulose acetate membrane functionalized with resveratrol exhibits a significant increase in alkaline phosphatase activity and extracellular matrix mineralization, suggesting its suitability to function as an implant surface coating for guided bone regeneration.

Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids

PubMed Central

Shvets, Elena; Bitsikas, Vassilis; Howard, Gillian; Hansen, Carsten Gram; Nichols, Benjamin J.

2015-01-01

Caveolae have long been implicated in endocytosis. Recent data question this link, and in the absence of specific cargoes the potential cellular function of caveolar endocytosis remains unclear. Here we develop new tools, including doubly genome-edited cell lines, to assay the subcellular dynamics of caveolae using tagged proteins expressed at endogenous levels. We find that around 5% of the cellular pool of caveolae is present on dynamic endosomes, and is delivered to endosomes in a clathrin-independent manner. Furthermore, we show that caveolae are indeed likely to bud directly from the plasma membrane. Using a genetically encoded tag for electron microscopy and ratiometric light microscopy, we go on to show that bulk membrane proteins are depleted within caveolae. Although caveolae are likely to account for only a small proportion of total endocytosis, cells lacking caveolae show fundamentally altered patterns of membrane traffic when loaded with excess glycosphingolipid. Altogether, these observations support the hypothesis that caveolar endocytosis is specialized for transport of membrane lipid. PMID:25897946

Non-enveloped virus reduction with quaternized chitosan nanofibers containing graphene.

PubMed

Bai, Bingyu; Mi, Xue; Xiang, Xu; Heiden, Patricia A; Heldt, Caryn L

2013-10-18

Membranes are an accepted technology for water purification. Membrane filtration can remove pathogens, including bacteria and viruses, by size. For small viruses that can have a diameter <25nm, removal by size leads to large membrane areas, high transmembrane pressures, low water flux, and frequent changing of membranes. In this work, we discovered that electrospun nanofibers made of chitosan and functionalized with a quaternary amine (HTCC) have the ability to adsorb a model non-enveloped virus, porcine parvovirus (PPV). To improve the virus removal of HTCC, we added graphene. Graphene both enhanced the ability to form nanofibers with HTCC and improved the virus removal. The hydrophobicity of graphene and the high charge of the HTCC create a system that can bind 95% of PPV. The HTCC/graphene nanofibers could be incorporated into microfiltration membranes and remove virus by adsorption. This would create a low pressure system that is more likely to benefit areas in need of fresh water. Copyright © 2013 Elsevier Ltd. All rights reserved.

Resolving mixed mechanisms of protein subdiffusion at the T cell plasma membrane

NASA Astrophysics Data System (ADS)

Golan, Yonatan; Sherman, Eilon

2017-06-01

The plasma membrane is a complex medium where transmembrane proteins diffuse and interact to facilitate cell function. Membrane protein mobility is affected by multiple mechanisms, including crowding, trapping, medium elasticity and structure, thus limiting our ability to distinguish them in intact cells. Here we characterize the mobility and organization of a short transmembrane protein at the plasma membrane of live T cells, using single particle tracking and photoactivated-localization microscopy. Protein mobility is highly heterogeneous, subdiffusive and ergodic-like. Using mobility characteristics, we segment individual trajectories into subpopulations with distinct Gaussian step-size distributions. Particles of low-to-medium mobility consist of clusters, diffusing in a viscoelastic and fractal-like medium and are enriched at the centre of the cell footprint. Particles of high mobility undergo weak confinement and are more evenly distributed. This study presents a methodological approach to resolve simultaneous mixed subdiffusion mechanisms acting on polydispersed samples and complex media such as cell membranes.

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

PubMed

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

2017-02-01

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

Ellagitannin HeT obtained from strawberry leaves is oxidized by bacterial membranes and inhibits the respiratory chain.

PubMed

Martos, Gustavo G; Mamani, Alicia; Filippone, María P; Abate, Pedro O; Katz, Néstor E; Castagnaro, Atilio P; Díaz Ricci, Juan C

2018-02-01

Plant secondary metabolism produces a variety of tannins that have a wide range of biological activities, including activation of plant defenses and antimicrobial, anti-inflammatory and antitumoral effects. The ellagitannin HeT (1- O -galloyl-2,3;4,6-bis-hexahydroxydiphenoyl-β-d-glucopyranose) from strawberry leaves elicits a strong plant defense response, and exhibits antimicrobial activity associated to the inhibition of the oxygen consumption, but its mechanism of action is unknown. In this paper we investigate the influence of HeT on bacterial cell membrane integrity and its effect on respiration. A β-galactosidase unmasking experiment showed that HeT does not disrupt membrane integrity. Raman spectroscopy analysis revealed that HeT strongly interacts with the cell membrane. Spectrochemical analysis indicated that HeT is oxidized in contact with bacterial cell membranes, and functional studies showed that HeT inhibits oxygen consumption, NADH and MTT reduction. These results provide evidence that HeT inhibits the respiratory chain.

Role of Flippases in Protein Glycosylation in the Endoplasmic Reticulum

PubMed Central

Rush, Jeffrey S.

2015-01-01

Glycosylation is essential to the synthesis, folding, and function of glycoproteins in eukaryotes. Proteins are co- and posttranslationally modified by a variety of glycans in the endoplasmic reticulum (ER); modifications include C- and O-mannosylation, N-glycosylation, and the addition of glycosylphosphatidylinositol membrane anchors. Protein glycosylation in the ER of eukaryotes involves enzymatic steps on both the cytosolic and lumenal surfaces of the ER membrane. The glycans are first assembled as precursor glycolipids, on the cytosolic surface of the ER, which are tethered to the membrane by attachment to a long-chain polyisoprenyl phosphate (dolichol) containing a reduced α-isoprene. The lipid-anchored building blocks then migrate transversely (flip) across the ER membrane to the lumenal surface, where final assembly of the glycan is completed. This strategy allows the cell to export high-energy biosynthetic intermediates as lipid-bound glycans, while constraining the glycosyl donors to the site of assembly on the membrane surface. This review focuses on the flippases that participate in protein glycosylation in the ER. PMID:26917968

Different sets of ER-resident J-proteins regulate distinct polar nuclear-membrane fusion events in Arabidopsis thaliana.

PubMed

Maruyama, Daisuke; Yamamoto, Masaya; Endo, Toshiya; Nishikawa, Shuh-ichi

2014-11-01

Angiosperm female gametophytes contain a central cell with two polar nuclei. In many species, including Arabidopsis thaliana, the polar nuclei fuse during female gametogenesis. We previously showed that BiP, an Hsp70 in the endoplasmic reticulum (ER), was essential for membrane fusion during female gametogenesis. Hsp70 function requires partner proteins for full activity. J-domain containing proteins (J-proteins) are the major Hsp70 functional partners. A. thaliana ER contains three soluble J-proteins, AtERdj3A, AtERdj3B, and AtP58(IPK). Here, we analyzed mutants of these proteins and determined that double-mutant ovules lacking AtP58(IPK) and AtERdj3A or AtERdj3B were defective in polar nuclear fusion. Electron microscopy analysis identified that polar nuclei were in close contact, but no membrane fusion occurred in mutant ovules lacking AtP58(IPK) and AtERdj3A. The polar nuclear outer membrane appeared to be connected via the ER remaining at the inner unfused membrane in mutant ovules lacking AtP58(IPK) and AtERdj3B. These results indicate that ER-resident J-proteins, AtP58(IPK)/AtERdj3A and AtP58(IPK)/AtERdj3B, function at distinct steps of polar nuclear-membrane fusion. Similar to the bip1 bip2 double mutant female gametophytes, the aterdj3a atp58(ipk) double mutant female gametophytes defective in fusion of the outer polar nuclear membrane displayed aberrant endosperm proliferation after fertilization with wild-type pollen. However, endosperm proliferated normally after fertilization of the aterdj3b atp58(ipk) double mutant female gametophytes defective in fusion of the inner membrane. Our results indicate that the polar nuclear fusion defect itself does not cause an endosperm proliferation defect. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

The Escherichia coli Cpx envelope stress response regulates genes of diverse function that impact antibiotic resistance and membrane integrity.

PubMed

Raivio, Tracy L; Leblanc, Shannon K D; Price, Nancy L

2013-06-01

The Cpx envelope stress response mediates adaptation to stresses that cause envelope protein misfolding. Adaptation is partly conferred through increased expression of protein folding and degradation factors. The Cpx response also plays a conserved role in the regulation of virulence determinant expression and impacts antibiotic resistance. We sought to identify adaptive mechanisms that may be involved in these important functions by characterizing changes in the transcriptome of two different Escherichia coli strains when the Cpx response is induced. We show that, while there is considerable strain- and condition-specific variability in the Cpx response, the regulon is enriched for proteins and functions that are inner membrane associated under all conditions. Genes that were changed by Cpx pathway induction under all conditions were involved in a number of cellular functions and included several intergenic regions, suggesting that posttranscriptional regulation is important during Cpx-mediated adaptation. Some Cpx-regulated genes are centrally involved in energetics and play a role in antibiotic resistance. We show that a number of small, uncharacterized envelope proteins are Cpx regulated and at least two of these affect phenotypes associated with membrane integrity. Altogether, our work suggests new mechanisms of Cpx-mediated envelope stress adaptation and antibiotic resistance.

Effect of supplemental taurine on juvenile channel catfish Ictalurus punctatus growth

USDA-ARS?s Scientific Manuscript database

Taurine is a beta-amino sulfur amino acid found in most animal tissues. It has many important biological functions in mammals including membrane stabilization, antioxidation, cellular osmoregulation, detoxification, neuromodulation, and brain and eye development. Taurine supplementation in juvenil...

Protein Composition of Trypanosoma brucei Mitochondrial Membranes

PubMed Central

Acestor, Nathalie; Panigrahi, Aswini K.; Ogata, Yuko; Anupama, Atashi; Stuart, Kenneth D.

2010-01-01

Mitochondria consist of four compartments, outer membrane, intermembrane space, inner membrane and matrix; each harboring specific functions and structures. In this study, we used mass spectrometry (LC-MS/MS) to characterize the protein composition of Trypanosoma brucei mitochondrial membranes, which were enriched by different biochemical fractionation techniques. The analyses identified 202 proteins that contain one or more transmembrane domain(s) and/or positive GRAVY scores. Of these, various criteria were used to assign 72 proteins to mitochondrial membranes with high confidence, and 106 with moderate to low confidence. The sub-cellular localization of a selected subset of 13 membrane assigned proteins was confirmed by tagging and immunofluorescence analysis. While most proteins assigned to mitochondrial membrane have putative roles in metabolic, energy generating, and transport processes, ~50% have no known function. These studies result in a comprehensive profile of the composition and sub-organellar location of proteins in the T. brucei mitochondrion thus, providing useful information on mitochondrial functions. PMID:19834910

Ion transport controlled by nanoparticle-functionalized membranes.

PubMed

Barry, Edward; McBride, Sean P; Jaeger, Heinrich M; Lin, Xiao-Min

2014-12-17

From proton exchange membranes in fuel cells to ion channels in biological membranes, the well-specified control of ionic interactions in confined geometries profoundly influences the transport and selectivity of porous materials. Here we outline a versatile new approach to control a membrane's electrostatic interactions with ions by depositing ligand-coated nanoparticles around the pore entrances. Leveraging the flexibility and control by which ligated nanoparticles can be synthesized, we demonstrate how ligand terminal groups such as methyl, carboxyl and amine can be used to tune the membrane charge density and control ion transport. Further functionality, exploiting the ligands as binding sites, is demonstrated for sulfonate groups resulting in an enhancement of the membrane charge density. We then extend these results to smaller dimensions by systematically varying the underlying pore diameter. As a whole, these results outline a previously unexplored method for the nanoparticle functionalization of membranes using ligated nanoparticles to control ion transport.

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

Electrochemical performance and transport properties of a Nafion membrane in a hydrogen-bromine cell environment

NASA Technical Reports Server (NTRS)

Baldwin, Richard S.

1987-01-01

The overall energy conversion efficiency of a hydrogen-bromine energy storage system is highly dependent upon the characteristics and performance of the ion-exchange membrane utilized as a half-cell separator. The electrochemical performance and transport properties of a duPont Nafion membrane in an aqueous HBr-Br2 environment were investigated. Membrane conductivity data are presented as a function of HBr concentration and temperature for the determination of ohmic voltage losses across the membrane in an operational cell. Diffusion-controlled bromine permeation rates and permeabilities are presented as functions of solution composition and temperature. Relationships between the degree of membrane hydration and the membrane transport characteristics are discussed. The solution chemistry of an operational hydrogen-bromine cell undergoing charge from 45% HBr to 5% HBr is discussed, and, based upon the experimentally observed bromine permeation behavior, predicted cell coulombic losses due to bromine diffusion through the membrane are presented as a function of the cell state-of-charge.

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.

Durable vesicles for reconstitution of membrane proteins in biotechnology.

PubMed

Beales, Paul A; Khan, Sanobar; Muench, Stephen P; Jeuken, Lars J C

2017-02-08

The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability. Amphiphilic block copolymers can also self-assemble into polymersomes: tough vesicles with improved stability compared with liposomes. This review discusses the reconstitution of membrane proteins into polymersomes and the more recent development of hybrid vesicles, which blend the robust nature of block copolymers with the biofunctionality of lipids. These novel synthetic vesicles hold great promise for enabling membrane proteins within biotechnologies by supporting their enhanced in vitro performance and could also contribute to fundamental biochemical and biophysical research by improving the stability of membrane proteins that are challenging to work with. © 2017 The Author(s).

TIMMDC1/C3orf1 functions as a membrane-embedded mitochondrial complex I assembly factor through association with the MCIA complex.

PubMed

Guarani, Virginia; Paulo, Joao; Zhai, Bo; Huttlin, Edward L; Gygi, Steven P; Harper, J Wade

2014-03-01

Complex I (CI) of the electron transport chain, a large membrane-embedded NADH dehydrogenase, couples electron transfer to the release of protons into the mitochondrial inner membrane space to promote ATP production through ATP synthase. In addition to being a central conduit for ATP production, CI activity has been linked to neurodegenerative disorders, including Parkinson's disease. CI is built in a stepwise fashion through the actions of several assembly factors. We employed interaction proteomics to interrogate the molecular associations of 15 core subunits and assembly factors previously linked to human CI deficiency, resulting in a network of 101 proteins and 335 interactions (edges). TIMMDC1, a predicted 4-pass membrane protein, reciprocally associated with multiple members of the MCIA CI assembly factor complex and core CI subunits and was localized in the mitochondrial inner membrane, and its depletion resulted in reduced CI activity and cellular respiration. Quantitative proteomics demonstrated a role for TIMMDC1 in assembly of membrane-embedded and soluble arms of the complex. This study defines a new membrane-embedded CI assembly factor and provides a resource for further analysis of CI biology.

Spatially Resolved Measurement of the Stress Tensor in Thin Membranes Using Bending Waves

NASA Astrophysics Data System (ADS)

Waitz, Reimar; Lutz, Carolin; Nößner, Stephan; Hertkorn, Michael; Scheer, Elke

2015-04-01

The mode shape of bending waves in thin silicon and silicon-carbide membranes is measured as a function of space and time, using a phase-shift interferometer with stroboscopic light. The mode shapes hold information about all the relevant mechanical parameters of the samples, including the spatial distribution of static prestress. We present a simple algorithm to obtain a map of the lateral tensor components of the prestress, with a spatial resolution much better than the wavelength of the bending waves. The method is not limited to measuring the stress of bending waves. It is applicable in almost any situation, where the fields determining the state of the system can be measured as a function of space and time.

Plant uncoupling mitochondrial proteins.

PubMed

Vercesi, Aníbal Eugênio; Borecký, Jiri; Maia, Ivan de Godoy; Arruda, Paulo; Cuccovia, Iolanda Midea; Chaimovich, Hernan

2006-01-01

Uncoupling proteins (UCPs) are membrane proteins that mediate purine nucleotide-sensitive free fatty acid-activated H(+) flux through the inner mitochondrial membrane. After the discovery of UCP in higher plants in 1995, it was acknowledged that these proteins are widely distributed in eukaryotic organisms. The widespread presence of UCPs in eukaryotes implies that these proteins may have functions other than thermogenesis. In this review, we describe the current knowledge of plant UCPs, including their discovery, biochemical properties, distribution, gene family, gene expression profiles, regulation of gene expression, and evolutionary aspects. Expression analyses and functional studies on the plant UCPs under normal and stressful conditions suggest that UCPs regulate energy metabolism in the cellular responses to stress through regulation of the electrochemical proton potential (Deltamu(H)+) and production of reactive oxygen species.

TRPV2.

PubMed

Kojima, Itaru; Nagasawa, Masahiro

2014-01-01

Transient receptor potential vanilloid type 2, TRPV2, is a calcium-permeable cation channel belonging to the TRPV channel family. This channel is activated by heat (>52 °C), various ligands, and mechanical stresses. In most of the cells, a large portion of TRPV2 is located in the endoplasmic reticulum under unstimulated conditions. Upon stimulation of the cells with phosphatidylinositol 3-kinase-activating ligands, TRPV2 is translocated to the plasma membrane and functions as a cation channel. Mechanical stress may also induce translocation of TRPV2 to the plasma membrane. The expression of TRPV2 is high in some types of cells including neurons, neuroendocrine cells, immune cells involved in innate immunity, and certain types of cancer cells. TRPV2 may modulate various cellular functions in these cells.

N-ethylmaleimide-sensitive factor interacts with the serotonin transporter and modulates its trafficking: implications for pathophysiology in autism

PubMed Central

2014-01-01

Background Changes in serotonin transporter (SERT) function have been implicated in autism. SERT function is influenced by the number of transporter molecules present at the cell surface, which is regulated by various cellular mechanisms including interactions with other proteins. Thus, we searched for novel SERT-binding proteins and investigated whether the expression of one such protein was affected in subjects with autism. Methods Novel SERT-binding proteins were examined by a pull-down system. Alterations of SERT function and membrane expression upon knockdown of the novel SERT-binding protein were studied in HEK293-hSERT cells. Endogenous interaction of SERT with the protein was evaluated in mouse brains. Alterations in the mRNA expression of SERT (SLC6A4) and the SERT-binding protein in the post-mortem brains and the lymphocytes of autism patients were compared to nonclinical controls. Results N-ethylmaleimide-sensitive factor (NSF) was identified as a novel SERT-binding protein. NSF was co-localized with SERT at the plasma membrane, and NSF knockdown resulted in decreased SERT expression at the cell membranes and decreased SERT uptake function. NSF was endogenously co-localized with SERT and interacted with SERT. While SLC6A4 expression was not significantly changed, NSF expression tended to be reduced in post-mortem brains, and was significantly reduced in lymphocytes of autistic subjects, which correlated with the severity of the clinical symptoms. Conclusions These data clearly show that NSF interacts with SERT under physiological conditions and is required for SERT membrane trafficking and uptake function. A possible role for NSF in the pathophysiology of autism through modulation of SERT trafficking, is suggested. PMID:24834316

Changes in topography and function of thylakoid membranes following membrane protein phosphorylation.

PubMed

Black, M T; Lee, P; Horton, P

1986-09-01

Changes in topography and function of pea (Pisum sativum L.) thylakoid membrane fractions following membrane protein phosphorylation have been studied. After protein phosphorylation the stromal membrane fraction had a higher chlorophyll a/b ratio, an increased content of light-harvesting chlorophyll protein and a higher ratio of chlorophyll to cytochrome f. This indicates that a pool of light-harvesting chlorophyll protein migrates from the photosystem II-enriched grana regions to the photosystem I-enriched stroma lamellae, in agreement with Kyle et al. (1984, Biochim. Biophys. Acta 765, 89-96) and Larsson et al. (1983, Eur. J. Biochem. 136, 25-29). Phosphorylation caused a stimulation in the rate of light-limited photosystem-I electron transfer in the unappressed membrane fraction, indicating that the translocated LHC-II becomes functionally associated with photosystem I.

Membrane preparation and solubilization.

PubMed

Roy, Ankita

2015-01-01

Membrane proteins play an essential role in several biological processes like ion transport, signal transduction, and electron transfer to name a few. For structural and functional studies of integral membrane proteins, it is critically important to isolate proteins from the membrane using biological detergents. Detergents disrupt the native lipid components of the native membrane and encase the membrane protein in an unnatural environment in aqueous solution. However, a particular membrane protein is best solubilized in a specific detergent; therefore, screening for the optimal detergent is essential. Apart from keeping the membrane protein monodispered in solution, the detergent has to be compatible with downstream processes to isolate and characterize a membrane protein. Over the past several years, a number of membrane proteins have been successfully isolated for structural and functional studies that allowed an outline of general strategies for isolating a novel membrane protein of interest. © 2015 Elsevier Inc. All rights reserved.

The structure and function of cell membranes examined by atomic force microscopy and single-molecule force spectroscopy.

PubMed

Shan, Yuping; Wang, Hongda

2015-06-07

The cell membrane is one of the most complicated biological complexes, and long-term fierce debates regarding the cell membrane persist because of technical hurdles. With the rapid development of nanotechnology and single-molecule techniques, our understanding of cell membranes has substantially increased. Atomic force microscopy (AFM) has provided several unprecedented advances (e.g., high resolution, three-dimensional and in situ measurements) in the study of cell membranes and has been used to systematically dissect the membrane structure in situ from both sides of membranes; as a result, novel models of cell membranes have recently been proposed. This review summarizes the new progress regarding membrane structure using in situ AFM and single-molecule force spectroscopy (SMFS), which may shed light on the study of the structure and functions of cell membranes.

Dual-Functional Ultrafiltration Membrane for Simultaneous Removal of Multiple Pollutants with High Performance.

PubMed

Pan, Shunlong; Li, Jiansheng; Noonan, Owen; Fang, Xiaofeng; Wan, Gaojie; Yu, Chengzhong; Wang, Lianjun

2017-05-02

Simultaneous removal of multiple pollutants from aqueous solution with less energy consumption is crucial in water purification. Here, a novel concept of dual-functional ultrafiltration (DFUF) membrane is demonstrated by entrapment of nanostructured adsorbents into the finger-like pores of ultrafiltration (UF) membrane rather than in the membrane matrix in previous reports of blend membranes, resulting in an exceptionally high active content and simultaneous removal of multiple pollutants from water due to the dual functions of rejection and adsorption. As a demonstration, hollow porous Zr(OH) x nanospheres (HPZNs) were immobilized in poly(ether sulfone) (PES) UF membranes through polydopamine coating with a high content of 68.9 wt %. The decontamination capacity of DFUF membranes toward multiple model pollutants (colloidal gold, polyethylene glycol (PEG), Pb(II)) was evaluated against a blend membrane. Compared to the blend membrane, the DFUF membranes showed 2.1-fold increase in the effective treatment volume for the treatment of Pb(II) contaminated water from 100 ppb to below 10 ppb (WHO drinking water standard). Simultaneously, the DFUF membranes effectively removed the colloidal gold and PEG below instrument detection limit, however the blend membrane only achieved 97.6% and 96.8% rejection for colloidal gold and PEG, respectively. Moreover, the DFUF membranes showed negligible leakage of nanoadsorbents during testing; and the membrane can be easily regenerated and reused. This study sheds new light on the design of high performance multifunction membranes for drinking water purification.

The Conserved ATPase Get3/Arr4 Modulates the Activity of Membrane-Associated Proteins in Saccharomyces cerevisiae

PubMed Central

Auld, Kathryn L.; Hitchcock, Amy L.; Doherty, Hugh K.; Frietze, Seth; Huang, Linda S.; Silver, Pamela A.

2006-01-01

The regulation of cellular membrane dynamics is crucial for maintaining proper cell growth and division. The Cdc48-Npl4-Ufd1 complex is required for several regulated membrane-associated processes as part of the ubiquitin–proteasome system, including ER-associated degradation and the control of lipid composition in yeast. In this study we report the results of a genetic screen in Saccharomyces cerevisiae for extragenic suppressors of a temperature-sensitive npl4 allele and the subsequent analysis of one suppressor, GET3/ARR4. The GET3 gene encodes an ATPase with homology to the regulatory component of the bacterial arsenic pump. Mutants of GET3 rescue several phenotypes of the npl4 mutant and transcription of GET3 is coregulated with the proteasome, illustrating a functional relationship between GET3 and NPL4 in the ubiquitin–proteasome system. We have further found that Get3 biochemically interacts with the trans-membrane domain proteins Get1/Mdm39 and Get2/Rmd7 and that Δget3 is able to suppress phenotypes of get1 and get2 mutants, including sporulation defects. In combination, our characterization of GET3 genetic and biochemical interactions with NPL4, GET1, and GET2 implicates Get3 in multiple membrane-dependent pathways. PMID:16816426

The role of membrane fluidization in the gel-assisted formation of giant polymersomes

DOE PAGES

Greene, Adrienne C.; Henderson, Ian M.; Gomez, Andrew; ...

2016-07-13

Polymersomes are being widely explored as synthetic analogs of lipid vesicles based on their enhanced stability and potential uses in a wide variety of applications in (e.g., drug delivery, cell analogs, etc.). Controlled formation of giant polymersomes for use in membrane studies and cell mimetic systems, however, is currently limited by low-yield production methodologies. Here, we describe for the first time, how the size distribution of giant poly(ethylene glycol)-poly(butadiene) (PEO-PBD) polymersomes formed by gel-assisted rehydration may be controlled based on membrane fluidization. We first show that the average diameter and size distribution of PEO-PBD polymersomes may be readily increased bymore » increasing the temperature of the rehydration solution. Further, we describe a correlative relationship between polymersome size and membrane fluidization through the addition of sucrose during rehydration, enabling the formation of PEO-PBD polymersomes with a range of diameters, including giant-sized vesicles (>100 μm). This correlative relationship suggests that sucrose may function as a small molecule fluidizer during rehydration, enhancing polymer diffusivity during formation and increasing polymersome size. Altogether the ability to easily regulate the size of PEO-PBD polymersomes based on membrane fluidity, either through temperature or fluidizers, has broadly applicability in areas including targeted therapeutic delivery and synthetic biology.« less

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.

Membrane nanodomains in plants: capturing form, function, and movement.

PubMed

Tapken, Wiebke; Murphy, Angus S

2015-03-01

The plasma membrane is the interface between the cell and the external environment. Plasma membrane lipids provide scaffolds for proteins and protein complexes that are involved in cell to cell communication, signal transduction, immune responses, and transport of small molecules. In animals, fungi, and plants, a substantial subset of these plasma membrane proteins function within ordered sterol- and sphingolipid-rich nanodomains. High-resolution microscopy, lipid dyes, pharmacological inhibitors of lipid biosynthesis, and lipid biosynthetic mutants have been employed to examine the relationship between the lipid environment and protein activity in plants. They have also been used to identify proteins associated with nanodomains and the pathways by which nanodomain-associated proteins are trafficked to their plasma membrane destinations. These studies suggest that plant membrane nanodomains function in a context-specific manner, analogous to similar structures in animals and fungi. In addition to the highly conserved flotillin and remorin markers, some members of the B and G subclasses of ATP binding cassette transporters have emerged as functional markers for plant nanodomains. Further, the glycophosphatidylinositol-anchored fasciclin-like arabinogalactan proteins, that are often associated with detergent-resistant membranes, appear also to have a functional role in membrane nanodomains. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Thermodynamics and Mechanics of Membrane Curvature Generation and Sensing by Proteins and Lipids

PubMed Central

Baumgart, Tobias; Capraro, Benjamin R.; Zhu, Chen; Das, Sovan L.

2014-01-01

Research investigating lipid membrane curvature generation and sensing is a rapidly developing frontier in membrane physical chemistry and biophysics. The fast recent progress is based on the discovery of a plethora of proteins involved in coupling membrane shape to cellular membrane function, the design of new quantitative experimental techniques to study aspects of membrane curvature, and the development of analytical theories and simulation techniques that allow a mechanistic interpretation of quantitative measurements. The present review first provides an overview of important classes of membrane proteins for which function is coupled to membrane curvature. We then survey several mechanisms that are assumed to underlie membrane curvature sensing and generation. Finally, we discuss relatively simple thermodynamic/mechanical models that allow quantitative interpretation of experimental observations. PMID:21219150

Hydroponics on a chip: analysis of the Fe deficient Arabidopsis thylakoid membrane proteome.

PubMed

Laganowsky, Arthur; Gómez, Stephen M; Whitelegge, Julian P; Nishio, John N

2009-04-13

The model plant Arabidopsis thaliana was used to evaluate the thylakoid membrane proteome under Fe-deficient conditions. Plants were cultivated using a novel hydroponic system, called "hydroponics on a chip", which yields highly reproducible plant tissue samples for physiological analyses, and can be easily used for in vivo stable isotope labeling. The thylakoid membrane proteome, from intact chloroplasts isolated from Fe-sufficient and Fe-deficient plants grown with hydroponics on a chip, was analyzed using liquid chromatography coupled to mass spectrometry. Intact masses of thylakoid membrane proteins were measured, many for the first time, and several proteins were identified with post-translational modifications that were altered by Fe deficiency; for example, the doubly phosphorylated form of the photosystem II oxygen evolving complex, PSBH, increased under Fe-deficiency. Increased levels of photosystem II protein subunit PSBS were detected in the Fe-deficient samples. Antioxidant enzymes, including ascorbate peroxidase and peroxiredoxin Q, were only detected in the Fe-deficient samples. We present the first biochemical evidence that the two major LHC IIb proteins (LHCB1 and LHCB2) may have significantly different functions in the thylakoid membrane. The study illustrates the utility of intact mass proteomics as an indispensable tool for functional genomics. "Hydroponics on a chip" provides the ability to grow A. thaliana under defined conditions that will be useful for systems biology.

Membranes for Periodontal Regeneration--A Materials Perspective.

PubMed

Bottino, Marco C; Thomas, Vinoy

2015-01-01

Periodontitis is a chronic inflammatory disorder affecting nearly 50% of adults in the United States. If left untreated, it can lead to the destruction of both soft and mineralized tissues that constitute the periodontium. Clinical management, including but not limited to flap debridement and/or curettage, as well as regenerative-based strategies with periodontal membranes associated or not with grafting materials, has been used with distinct levels of success. Unquestionably, no single implantable biomaterial can consistently guide the coordinated growth and development of multiple tissue types, especially in very large periodontal defects. With the global aging population, it is extremely important to find novel biomaterials, particularly bioactive membranes and/or scaffolds, for guided tissue (GTR) and bone regeneration (GBR) to aid in the reestablishment of the health and function of distinct periodontal tissues. This chapter offers an update on the evolution of biomaterials (i.e. membranes and bioactive scaffolds) as well as material-based strategies applied in periodontal regeneration. The authors start by providing a brief summary of the histological characteristics and functions of the periodontium and its main pathological condition, namely periodontitis. Next, a review of commercially available GTR/GBR membranes is given, followed by a critical appraisal of the most recent advances in the development of bioactive materials that enhance the chance for clinical success of periodontal tissue regeneration. © 2015 S. Karger AG, Basel.

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

PubMed

Halova, Ivana; Draber, Petr

2016-01-01

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

Atomistic models for free energy evaluation of drug binding to membrane proteins.

PubMed

Durdagi, S; Zhao, C; Cuervo, J E; Noskov, S Y

2011-01-01

The binding of various molecules to integral membrane proteins with optimal affinity and specificity is central to normal function of cell. While membrane proteins represent about one third of the whole cell proteome, they are a majority of common drug targets. The quest for the development of computational models capable of accurate evaluation of binding affinities, decomposition of the binding into its principal components and thus mapping molecular mechanisms of binding remains one of the main goals of modern computational biophysics and related drug development. The primary scope of this review will be on the recent extension of computational methods for the study of drug binding to membrane proteins. Several examples of such applications will be provided ranging from secondary transporters to voltage gated channels. In this mini-review, we will provide a short summary on the breadth of different methods for binding affinity evaluation. These methods include molecular docking with docking scoring functions, molecular dynamics (MD) simulations combined with post-processing analysis using Molecular Mechanics/Poisson Boltzmann (Generalized Born) Surface Area (MM/PB(GB)SA), as well as direct evaluation of free energies from Free Energy Perturbation (FEP) with constraining schemes, and Potential of Mean Force (PMF) computations. We will compare advantages and shortcomings of popular techniques and provide discussion on the integrative strategies for drug development aimed at targeting membrane proteins.

Mitigation of Biofilm Development on Thin-Film Composite Membranes Functionalized with Zwitterionic Polymers and Silver Nanoparticles.

PubMed

Liu, Caihong; Faria, Andreia F; Ma, Jun; Elimelech, Menachem

2017-01-03

We demonstrate the functionalization of thin-film composite membranes with zwitterionic polymers and silver nanoparticles (AgNPs) for combating biofouling. Combining hydrophilic zwitterionic polymer brushes and biocidal AgNPs endows the membrane with dual functionality: antiadhesion and bacterial inactivation. An atom transfer radical polymerization (ATRP) reaction is used to graft zwitterionic poly(sulfobetaine methacrylate) (PSBMA) brushes to the membrane surface, while AgNPs are synthesized in situ through chemical reduction of silver. Two different membrane architectures (Ag-PSBMA and PSBMA-Ag TFC) are developed according to the sequence AgNPs, and PSBMA brushes are grafted on the membrane surface. A static adhesion assay shows that both modified membranes significantly reduced the adsorption of proteins, which served as a model organic foulant. However, improved antimicrobial activity is observed for PSBMA-Ag TFC (i.e., AgNPs on top of the polymer brush) in comparison to the Ag-PSBMA TFC membrane (i.e., polymer brush on top of AgNPs), indicating that architecture of the antifouling layer is an important factor in the design of zwitterion-silver membranes. Confocal laser scanning microscopy (CLSM) imaging indicated that PSBMA-Ag TFC membranes effectively inhibit biofilm formation under dynamic cross-flow membrane biofouling tests. Finally, we demonstrate the regeneration of AgNPs on the membrane after depletion of silver from the surface of the PSBMA-Ag TFC membrane.

Oral supplementation with docosahexaenoic acid and uridine-5'-monophosphate increases dendritic spine density in adult gerbil hippocampus.

PubMed

Sakamoto, Toshimasa; Cansev, Mehmet; Wurtman, Richard J

2007-11-28

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is an essential component of membrane phosphatides and has been implicated in cognitive functions. Low levels of circulating or brain DHA are associated with various neurocognitive disorders including Alzheimer's disease (AD), while laboratory animals, including animal models of AD, can exhibit improved cognitive ability with a diet enriched in DHA. Various cellular mechanisms have been proposed for DHA's behavioral effects, including increases in cellular membrane fluidity, promotion of neurite extension and inhibition of apoptosis. However, there is little direct evidence that DHA affects synaptic structure in living animals. Here we show that oral supplementation with DHA substantially increases the number of dendritic spines in adult gerbil hippocampus, particularly when animals are co-supplemented with a uridine source, uridine-5'-monophosphate (UMP), which increases brain levels of the rate-limiting phosphatide precursor CTP. The increase in dendritic spines (>30%) is accompanied by parallel increases in membrane phosphatides and in pre- and post-synaptic proteins within the hippocampus. Hence, oral DHA may promote neuronal membrane synthesis to increase the number of synapses, particularly when co-administered with UMP. Our findings provide a possible explanation for the effects of DHA on behavior and also suggest a strategy to treat cognitive disorders resulting from synapse loss.

Pulmonary Vascular Congestion: A Mechanism for Distal Lung Unit Dysfunction in Obesity.

PubMed

Oppenheimer, Beno W; Berger, Kenneth I; Ali, Saleem; Segal, Leopoldo N; Donnino, Robert; Katz, Stuart; Parikh, Manish; Goldring, Roberta M

2016-01-01

Obesity is characterized by increased systemic and pulmonary blood volumes (pulmonary vascular congestion). Concomitant abnormal alveolar membrane diffusion suggests subclinical interstitial edema. In this setting, functional abnormalities should encompass the entire distal lung including the airways. We hypothesize that in obesity: 1) pulmonary vascular congestion will affect the distal lung unit with concordant alveolar membrane and distal airway abnormalities; and 2) the degree of pulmonary congestion and membrane dysfunction will relate to the cardiac response. 54 non-smoking obese subjects underwent spirometry, impulse oscillometry (IOS), diffusion capacity (DLCO) with partition into membrane diffusion (DM) and capillary blood volume (VC), and cardiac MRI (n = 24). Alveolar-capillary membrane efficiency was assessed by calculation of DM/VC. Mean age was 45±12 years; mean BMI was 44.8±7 kg/m2. Vital capacity was 88±13% predicted with reduction in functional residual capacity (58±12% predicted). Despite normal DLCO (98±18% predicted), VC was elevated (135±31% predicted) while DM averaged 94±22% predicted. DM/VC varied from 0.4 to 1.4 with high values reflecting recruitment of alveolar membrane and low values indicating alveolar membrane dysfunction. The most abnormal IOS (R5 and X5) occurred in subjects with lowest DM/VC (r2 = 0.31, p<0.001; r2 = 0.34, p<0.001). Cardiac output and index (cardiac output / body surface area) were directly related to DM/VC (r2 = 0.41, p<0.001; r2 = 0.19, p = 0.03). Subjects with lower DM/VC demonstrated a cardiac output that remained in the normal range despite presence of obesity. Global dysfunction of the distal lung (alveolar membrane and distal airway) is associated with pulmonary vascular congestion and failure to achieve the high output state of obesity. Pulmonary vascular congestion and consequent fluid transudation and/or alterations in the structure of the alveolar capillary membrane may be considered often unrecognized causes of airway dysfunction in obesity.

Role of DHA in aging-related changes in mouse brain synaptic plasma membrane proteome.

PubMed

Sidhu, Vishaldeep K; Huang, Bill X; Desai, Abhishek; Kevala, Karl; Kim, Hee-Yong

2016-05-01

Aging has been related to diminished cognitive function, which could be a result of ineffective synaptic function. We have previously shown that synaptic plasma membrane proteins supporting synaptic integrity and neurotransmission were downregulated in docosahexaenoic acid (DHA)-deprived brains, suggesting an important role of DHA in synaptic function. In this study, we demonstrate aging-induced synaptic proteome changes and DHA-dependent mitigation of such changes using mass spectrometry-based protein quantitation combined with western blot or messenger RNA analysis. We found significant reduction of 15 synaptic plasma membrane proteins in aging brains including fodrin-α, synaptopodin, postsynaptic density protein 95, synaptic vesicle glycoprotein 2B, synaptosomal-associated protein 25, synaptosomal-associated protein-α, N-methyl-D-aspartate receptor subunit epsilon-2 precursor, AMPA2, AP2, VGluT1, munc18-1, dynamin-1, vesicle-associated membrane protein 2, rab3A, and EAAT1, most of which are involved in synaptic transmission. Notably, the first 9 proteins were further reduced when brain DHA was depleted by diet, indicating that DHA plays an important role in sustaining these synaptic proteins downregulated during aging. Reduction of 2 of these proteins was reversed by raising the brain DHA level by supplementing aged animals with an omega-3 fatty acid sufficient diet for 2 months. The recognition memory compromised in DHA-depleted animals was also improved. Our results suggest a potential role of DHA in alleviating aging-associated cognitive decline by offsetting the loss of neurotransmission-regulating synaptic proteins involved in synaptic function. Published by Elsevier Inc.

Foulant Analysis of Three RO Membranes Used in Treating Simulated Brackish Water of the Iraqi Marshes

PubMed Central

Sachit, Dawood Eisa; Veenstra, John N.

2017-01-01

In this work, three different types of Reverse Osmosis (RO) (Thin-Film Composite (SE), Cellulose Acetate (CE), and Polyamide (AD)) were used to perform foulant analysis (autopsy) study on the deposited materials from three different simulated brackish surface feed waters. The brackish surface water qualities represented the water quality in Iraqi marshes. The main foulants from the simulated feed waters were characterized by using Scanning Electron Microscope (SEM) images and Energy-Dispersive X-ray Spectroscopy (EDXS) spectra. The effect of feed water temperatures (37 °C and 11 °C) on the formation of the fouled material deposited on the membrane surface was examined in this study. Also, pretreatment by a 0.1 micron microfiltration (MF) membrane of the simulated feed water in advance of the RO membrane on the precipitated material on the membrane surface was investigated. Finally, Fourier Transform Infrared Spectroscopy (FTIR) analysis was used to identify the functional groups of the organic matter deposited on the RO membrane surfaces. The SEM images and EDSX spectra suggested that the fouled material was mainly organic matter, and the major crystal deposited on the RO membrane was calcium carbonate (CaCO3). The FTIR spectra of the fouled RO membranes suggested that the constituents of the fouled material included aliphatic and aromatic compounds. PMID:28406468

Reversible Self-Actuated Thermo-Responsive Pore Membrane

PubMed Central

Park, Younggeun; Gutierrez, Maria Paz; Lee, Luke P.

2016-01-01

Smart membranes, which can selectively control the transfer of light, air, humidity and temperature, are important to achieve indoor climate regulation. Even though reversible self-actuation of smart membranes is desirable in large-scale, reversible self-regulation remains challenging. Specifically, reversible 100% opening/closing of pore actuation showing accurate responsiveness, reproducibility and structural flexibility, including uniform structure assembly, is currently very difficult. Here, we report a reversible, thermo-responsive self-activated pore membrane that achieves opening and closing of pores. The reversible, self-actuated thermo-responsive pore membrane was fabricated with hybrid materials of poly (N-isopropylacrylamide), (PNIPAM) within polytetrafluoroethylene (PTFE) to form a multi-dimensional pore array. Using Multiphysics simulation of heat transfer and structural mechanics based on finite element analysis, we demonstrated that pore opening and closing dynamics can be self-activated at environmentally relevant temperatures. Temperature cycle characterizations of the pore structure revealed 100% opening ratio at T = 40 °C and 0% opening ratio at T = 20 °C. The flexibility of the membrane showed an accurate temperature-responsive function at a maximum bending angle of 45°. Addressing the importance of self-regulation, this reversible self-actuated thermo-responsive pore membrane will advance the development of future large-scale smart membranes needed for sustainable indoor climate control. PMID:27991563

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.

Reversible Self-Actuated Thermo-Responsive Pore Membrane

NASA Astrophysics Data System (ADS)

Park, Younggeun; Gutierrez, Maria Paz; Lee, Luke P.

2016-12-01

Smart membranes, which can selectively control the transfer of light, air, humidity and temperature, are important to achieve indoor climate regulation. Even though reversible self-actuation of smart membranes is desirable in large-scale, reversible self-regulation remains challenging. Specifically, reversible 100% opening/closing of pore actuation showing accurate responsiveness, reproducibility and structural flexibility, including uniform structure assembly, is currently very difficult. Here, we report a reversible, thermo-responsive self-activated pore membrane that achieves opening and closing of pores. The reversible, self-actuated thermo-responsive pore membrane was fabricated with hybrid materials of poly (N-isopropylacrylamide), (PNIPAM) within polytetrafluoroethylene (PTFE) to form a multi-dimensional pore array. Using Multiphysics simulation of heat transfer and structural mechanics based on finite element analysis, we demonstrated that pore opening and closing dynamics can be self-activated at environmentally relevant temperatures. Temperature cycle characterizations of the pore structure revealed 100% opening ratio at T = 40 °C and 0% opening ratio at T = 20 °C. The flexibility of the membrane showed an accurate temperature-responsive function at a maximum bending angle of 45°. Addressing the importance of self-regulation, this reversible self-actuated thermo-responsive pore membrane will advance the development of future large-scale smart membranes needed for sustainable indoor climate control.

N-3 fatty acids and membrane microdomains: from model membranes to lymphocyte function.

PubMed

Shaikh, Saame Raza; Teague, Heather

2012-12-01

This article summarizes the author's research on fish oil derived n-3 fatty acids, plasma membrane organization and B cell function. We first cover basic model membrane studies that investigated how docosahexaenoic acid (DHA) targeted the organization of sphingolipid-cholesterol enriched lipid microdomains. A key finding here was that DHA had a relatively poor affinity for cholesterol. This work led to a model that predicted DHA acyl chains in cells would manipulate lipid-protein microdomain organization and thereby function. We then review how the predictions of the model were tested with B cells in vitro followed by experiments using mice fed fish oil. These studies reveal a highly complex picture on how n-3 fatty acids target lipid-protein organization and B cell function. Key findings are as follows: (1) n-3 fatty acids target not just the plasma membrane but also endomembrane organization; (2) DHA, but not eicosapentaenoic acid (EPA), disrupts microdomain spatial distribution (i.e. clustering), (3) DHA alters protein lateral organization and (4) changes in membrane organization are accompanied by functional effects on both innate and adaptive B cell function. Altogether, the research over the past 10 years has led to an evolution of the original model on how DHA reorganizes membrane microdomains. The work raises the intriguing possibility of testing the model at the human level to target health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

YTPdb: a wiki database of yeast membrane transporters.

PubMed

Brohée, Sylvain; Barriot, Roland; Moreau, Yves; André, Bruno

2010-10-01

Membrane transporters constitute one of the largest functional categories of proteins in all organisms. In the yeast Saccharomyces cerevisiae, this represents about 300 proteins ( approximately 5% of the proteome). We here present the Yeast Transport Protein database (YTPdb), a user-friendly collaborative resource dedicated to the precise classification and annotation of yeast transporters. YTPdb exploits an evolution of the MediaWiki web engine used for popular collaborative databases like Wikipedia, allowing every registered user to edit the data in a user-friendly manner. Proteins in YTPdb are classified on the basis of functional criteria such as subcellular location or their substrate compounds. These classifications are hierarchical, allowing queries to be performed at various levels, from highly specific (e.g. ammonium as a substrate or the vacuole as a location) to broader (e.g. cation as a substrate or inner membranes as location). Other resources accessible for each transporter via YTPdb include post-translational modifications, K(m) values, a permanently updated bibliography, and a hierarchical classification into families. The YTPdb concept can be extrapolated to other organisms and could even be applied for other functional categories of proteins. YTPdb is accessible at http://homes.esat.kuleuven.be/ytpdb/. Copyright © 2010 Elsevier B.V. All rights reserved.

Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

PubMed

Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

2014-01-01

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress

PubMed Central

Pascua-Maestro, Raquel

2017-01-01

Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular functions, critical for the outcome of a wide variety of neurodegenerative diseases. These results open therapeutic opportunities by providing a route of entry and a repair mechanism for lysosomes in pathological situations. PMID:28182653

Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays.

PubMed

Ghosh, Madhubrata; Wang, Loo Chien; Ramesh, Ranita; Morgan, Leslie K; Kenney, Linda J; Anand, Ganesh S

2017-02-28

Membrane-anchored receptors are essential cellular signaling elements for stimulus sensing, propagation, and transmission inside cells. However, the contributions of lipid interactions to the function and dynamics of embedded receptor kinases have not been described in detail. In this study, we used amide hydrogen/deuterium exchange mass spectrometry, a sensitive biophysical approach, to probe the dynamics of a membrane-embedded receptor kinase, EnvZ, together with functional assays to describe the role of lipids in receptor kinase function. Our results reveal that lipids play an important role in regulating receptor function through interactions with transmembrane segments, as well as through peripheral interactions with nonembedded domains. Specifically, the lipid membrane allosterically modulates the activity of the embedded kinase by altering the dynamics of a glycine-rich motif that is critical for phosphotransfer from ATP. This allostery in EnvZ is independent of membrane composition and involves direct interactions with transmembrane and periplasmic segments, as well as peripheral interactions with nonembedded domains of the protein. In the absence of the membrane-spanning regions, lipid allostery is propagated entirely through peripheral interactions. Whereas lipid allostery impacts the phosphotransferase function of the kinase, extracellular stimulus recognition is mediated via a four-helix bundle subdomain located in the cytoplasm, which functions as the osmosensing core through osmolality-dependent helical stabilization. Our findings emphasize the functional modularity in a membrane-embedded kinase, separated into membrane association, phosphotransferase function, and stimulus recognition. These components are integrated through long-range communication relays, with lipids playing an essential role in regulation. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Butterfly gyroid nanostructures as a time-frozen glimpse of intracellular membrane development

PubMed Central

Wilts, Bodo D.; Apeleo Zubiri, Benjamin; Klatt, Michael A.; Butz, Benjamin; Fischer, Michael G.; Kelly, Stephen T.; Spiecker, Erdmann; Steiner, Ullrich; Schröder-Turk, Gerd E.

2017-01-01

The formation of the biophotonic gyroid material in butterfly wing scales is an exceptional feat of evolutionary engineering of functional nanostructures. It is hypothesized that this nanostructure forms by chitin polymerization inside a convoluted membrane of corresponding shape in the endoplasmic reticulum. However, this dynamic formation process, including whether membrane folding and chitin expression are simultaneous or sequential processes, cannot yet be elucidated by in vivo imaging. We report an unusual hierarchical ultrastructure in the butterfly Thecla opisena that, as a solid material, allows high-resolution three-dimensional microscopy. Rather than the conventional polycrystalline space-filling arrangement, a gyroid occurs in isolated facetted crystallites with a pronounced size gradient. When interpreted as a sequence of time-frozen snapshots of the morphogenesis, this arrangement provides insight into the formation mechanisms of the nanoporous gyroid material as well as of the intracellular organelle membrane that acts as the template. PMID:28508050

Peptide-Lipid Interactions: Experiments and Applications

PubMed Central

Galdiero, Stefania; Falanga, Annarita; Cantisani, Marco; Vitiello, Mariateresa; Morelli, Giancarlo; Galdiero, Massimiliano

2013-01-01

The interactions between peptides and lipids are of fundamental importance in the functioning of numerous membrane-mediated cellular processes including antimicrobial peptide action, hormone-receptor interactions, drug bioavailability across the blood-brain barrier and viral fusion processes. Moreover, a major goal of modern biotechnology is obtaining new potent pharmaceutical agents whose biological action is dependent on the binding of peptides to lipid-bilayers. Several issues need to be addressed such as secondary structure, orientation, oligomerization and localization inside the membrane. At the same time, the structural effects which the peptides cause on the lipid bilayer are important for the interactions and need to be elucidated. The structural characterization of membrane active peptides in membranes is a harsh experimental challenge. It is in fact accepted that no single experimental technique can give a complete structural picture of the interaction, but rather a combination of different techniques is necessary. PMID:24036440

Ultra-thin and strong formvar-based membranes with controlled porosity for micro- and nano-scale systems

DOE PAGES

Auchter, Eric Lawrence; Marquez, Justin Ryan; Stevens, Garrison Nicole; ...

2018-03-28

Here, we present a methodology for developing ultra-thin and strong formvar-based membranes with controlled morphologies. Formvar is a thin hydrophilic and oleophilic polymer inert to most chemicals and resistant to radiation. The formvar-based membranes are viable materials as support structures in micro- and macro-scale systems depending on thinness and porosity control. Tunable sub-micron thick porous membranes with 20%–65% porosity were synthesized by controlling the ratios of formvar, glycerol, and chloroform. This synthesis process does not require complex separation or handling methods and allows for the production of strong, thin, and porous formvar-based membranes. An expansive array of these membrane characterizationsmore » including chemical compatibility, mechanical responses, wettability, as well as the mathematical simulations as a function of porosity has been presented. The wide range of chemical compatibility allows for membrane applications in various environments, where other polymers would not be suitable. Our formvar-based membranes were found to have an elastic modulus of 7.8 GPa, a surface free energy of 50 mN m -1 and an average thickness of 125 nm. Stochastic model simulations indicate that formvar with the porosity of ~50% is the optimal membrane formulation, allowing the most material transfer across the membrane while also withstanding the highest simulated pressure loadings before tearing. Development of novel, resilient and versatile membranes with controlled porosity offers a wide range of exciting applications in the fields of nanoscience, microfluidics, and MEMS.« less

Ultra-thin and strong formvar-based membranes with controlled porosity for micro- and nano-scale systems

NASA Astrophysics Data System (ADS)

Auchter, Eric; Marquez, Justin; Stevens, Garrison; Silva, Rebecca; Mcculloch, Quinn; Guengerich, Quintessa; Blair, Andrew; Litchfield, Sebastian; Li, Nan; Sheehan, Chris; Chamberlin, Rebecca; Yarbro, Stephen L.; Dervishi, Enkeleda

2018-05-01

We present a methodology for developing ultra-thin and strong formvar-based membranes with controlled morphologies. Formvar is a thin hydrophilic and oleophilic polymer inert to most chemicals and resistant to radiation. The formvar-based membranes are viable materials as support structures in micro- and macro-scale systems depending on thinness and porosity control. Tunable sub-micron thick porous membranes with 20%–65% porosity were synthesized by controlling the ratios of formvar, glycerol, and chloroform. This synthesis process does not require complex separation or handling methods and allows for the production of strong, thin, and porous formvar-based membranes. An expansive array of these membrane characterizations including chemical compatibility, mechanical responses, wettability, as well as the mathematical simulations as a function of porosity has been presented. The wide range of chemical compatibility allows for membrane applications in various environments, where other polymers would not be suitable. Our formvar-based membranes were found to have an elastic modulus of 7.8 GPa, a surface free energy of 50 mN m‑1 and an average thickness of 125 nm. Stochastic model simulations indicate that formvar with the porosity of ∼50% is the optimal membrane formulation, allowing the most material transfer across the membrane while also withstanding the highest simulated pressure loadings before tearing. Development of novel, resilient and versatile membranes with controlled porosity offers a wide range of exciting applications in the fields of nanoscience, microfluidics, and MEMS.

Ultra-thin and strong formvar-based membranes with controlled porosity for micro- and nano-scale systems

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

Auchter, Eric Lawrence; Marquez, Justin Ryan; Stevens, Garrison Nicole

Here, we present a methodology for developing ultra-thin and strong formvar-based membranes with controlled morphologies. Formvar is a thin hydrophilic and oleophilic polymer inert to most chemicals and resistant to radiation. The formvar-based membranes are viable materials as support structures in micro- and macro-scale systems depending on thinness and porosity control. Tunable sub-micron thick porous membranes with 20%–65% porosity were synthesized by controlling the ratios of formvar, glycerol, and chloroform. This synthesis process does not require complex separation or handling methods and allows for the production of strong, thin, and porous formvar-based membranes. An expansive array of these membrane characterizationsmore » including chemical compatibility, mechanical responses, wettability, as well as the mathematical simulations as a function of porosity has been presented. The wide range of chemical compatibility allows for membrane applications in various environments, where other polymers would not be suitable. Our formvar-based membranes were found to have an elastic modulus of 7.8 GPa, a surface free energy of 50 mN m -1 and an average thickness of 125 nm. Stochastic model simulations indicate that formvar with the porosity of ~50% is the optimal membrane formulation, allowing the most material transfer across the membrane while also withstanding the highest simulated pressure loadings before tearing. Development of novel, resilient and versatile membranes with controlled porosity offers a wide range of exciting applications in the fields of nanoscience, microfluidics, and MEMS.« less

Effects of Polymer Structure and Relaxations on Ionic Conductivity in Anion Exchange Membranes with Quaternary Ammonium Functional Groups

NASA Astrophysics Data System (ADS)

Maes, Ashley M.

Anion exchange membranes (AEMs) are of considerable interest to developers and researchers of electrochemical conversion and storage devices such as anion exchange membrane fuel cells (AAEMFCs), alkaline polymer electrolyte electrolysers, redox flow batteries and bioelectrochemical devices. AEMs are generally in competition with more established proton exchange membranes (PEMs), but offer the potential for reduction of materials costs and greater fuel flexibility across these applications. This work includes an introduction to AEMs in the context of fuel cell technologies and some key techniques for AEM characterization. There are many synthetic strategies to incorporate cationic functional groups, which promote anion transport, into a polymer matrix. Two membrane chemistries are investigated in the following chapters. The first is based on a simple synthesis procedure that produced a membrane consisting of random, crosslinked polypropylene- ran-polyethyleneimine with quaternary ammonium functional groups. This membrane had moderate chloride ionic conductivity of 0.03 S cm -1 at 95 °C and high water uptake with minimal dimensional swelling. However, the lack of control of crosslink location and density during synthesis produced a material with a very random nature, making it a poor candidate for more fundamental transport studies. The second membrane chemistry is a block copolymer with a hydrophobic and hydrophilic block. The hydrophobic block was selected to provide favorable mechanical and barrier characteristics while a hydrophilic block was selected to provide water uptake and anion conducting functionalities. Poly(vinyl benzyl trimethyl ammonium bromide)-b-poly(methylbutylene) ([PVBTMA][Br]- b-PMB) was synthesized by partners at the University of Massachusetts-Amherst with varied degrees of functionalization (DF) along the hydrophilic block, resulting in ion exchange capacities ranging from 0.77 to 2.20 mmol g -1. Water uptake, in-plane ionic conductivity and membrane morphology were measured across a series of membranes with the original bromide (Br -) counter-ion. These bulk materials characterization experiments demonstrated that this polymer structure produces well-ordered lamellar morphology with moderate water uptake and competitive ionic conductivity (ca. 40 mS cm-1 at 90 °C and 95% relative humidity). These characteristics make it an appropriate candidate for the following more fundamental investigations of ionic conductivity mechanisms. Broadband electrical spectroscopy (BES) was conducted on one [PVBTMA][Br]- b-PMB sample in the Br- form and analyzed in conjunction with thermal stability and relaxation experiments in Chapter 4. We were able to propose two separate ionic conductivity mechanisms and relate each to physical attributes of the polymer structure. A significant thermal transition was observed at Tdelta , which resulted in a dramatic drop in conductivity. In a continued effort to characterize the ionic conductivity of these block-copolymer membranes, another BES study was conducted on three samples with varying DFs. Samples were converted to hydroxide (OH- ) form so we could contrast the Br- conductivity mechanisms to those in a more relevant counter-ion form. After analysis of the electric response of the material, combined with the thermal analysis by TGA, MDSC and DMA, conductivity mechanisms were described. As in the Br- study, conductivity involves two distinct conduction pathways, sigmaEP and sigmaIP,1. Importantly, we again observed a drop in conductivity at Tdelta in each of these samples, with Tdelta decreasing as the density of functional groups along the hydrophilic block increased. It is undesirable for this transition to occur during operation in a fuel cell or other electrochemical device, so future work to investigate strategies for inhibition are recommended.

Separation of Biologically Active Compounds by Membrane Operations.

PubMed

Zhu, Xiaoying; Bai, Renbi

2017-01-01

Bioactive compounds from various natural sources have been attracting more and more attention, owing to their broad diversity of functionalities and availabilities. However, many of the bioactive compounds often exist at an extremely low concentration in a mixture so that massive harvesting is needed to obtain sufficient amounts for their practical usage. Thus, effective fractionation or separation technologies are essential for the screening and production of the bioactive compound products. The applicatons of conventional processes such as extraction, distillation and lyophilisation, etc. may be tedious, have high energy consumption or cause denature or degradation of the bioactive compounds. Membrane separation processes operate at ambient temperature, without the need for heating and therefore with less energy consumption. The "cold" separation technology also prevents the possible degradation of the bioactive compounds. The separation process is mainly physical and both fractions (permeate and retentate) of the membrane processes may be recovered. Thus, using membrane separation technology is a promising approach to concentrate and separate bioactive compounds. A comprehensive survey of membrane operations used for the separation of bioactive compounds is conducted. The available and established membrane separation processes are introduced and reviewed. The most frequently used membrane processes are the pressure driven ones, including microfiltration (MF), ultrafiltration (UF) and nanofiltration (NF). They are applied either individually as a single sieve or in combination as an integrated membrane array to meet the different requirements in the separation of bioactive compounds. Other new membrane processes with multiple functions have also been developed and employed for the separation or fractionation of bioactive compounds. The hybrid electrodialysis (ED)-UF membrane process, for example has been used to provide a solution for the separation of biomolecules with similar molecular weights but different surface electrical properties. In contrast, the affinity membrane technology is shown to have the advantages of increasing the separation efficiency at low operational pressures through selectively adsorbing bioactive compounds during the filtration process. Individual membranes or membrane arrays are effectively used to separate bioactive compounds or achieve multiple fractionation of them with different molecule weights or sizes. Pressure driven membrane processes are highly efficient and widely used. Membrane fouling, especially irreversible organic and biological fouling, is the inevitable problem. Multifunctional membranes and affinity membranes provide the possibility of effectively separating bioactive compounds that are similar in sizes but different in other physical and chemical properties. Surface modification methods are of great potential to increase membrane separation efficiency as well as reduce the problem of membrane fouling. Developing membranes and optimizing the operational parameters specifically for the applications of separation of various bioactive compounds should be taken as an important part of ongoing or future membrane research in this field. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Microdosimetric study for nanosecond pulsed electric fields on a cell circuit model with nucleus.

PubMed

Denzi, Agnese; Merla, Caterina; Camilleri, Paola; Paffi, Alessandra; d'Inzeo, Guglielmo; Apollonio, Francesca; Liberti, Micaela

2013-10-01

Recently, scientific interest in electric pulses, always more intense and shorter and able to induce biological effects on both plasma and nuclear membranes, has greatly increased. Hence, microdosimetric models that include internal organelles like the nucleus have assumed increasing importance. In this work, a circuit model of the cell including the nucleus is proposed, which accounts for the dielectric dispersion of all cell compartments. The setup of the dielectric model of the nucleus is of fundamental importance in determining the transmembrane potential (TMP) induced on the nuclear membrane; here, this is demonstrated by comparing results for three different sets of nuclear dielectric properties present in the literature. The results have been compared, even including or disregarding the dielectric dispersion of the nucleus. The main differences have been found when using pulses shorter than 10 ns. This is due to the fact that the high spectral components of the shortest pulses are differently taken into account by the nuclear membrane transfer functions computed with and without nuclear dielectric dispersion. The shortest pulses are also the most effective in porating the intracellular structures, as confirmed by the time courses of the TMP calculated across the plasma and nuclear membranes. We show how dispersive nucleus models are unavoidable when dealing with pulses shorter than 10 ns because of the large spectral contents arriving above 100 MHz, i.e., over the typical relaxation frequencies of the dipolar mechanism of the molecules constituting the nuclear membrane and the subcellular cell compartments.

The mechanism of protein export enhancement by the SecDF membrane component

PubMed Central

Tsukazaki, Tomoya; Nureki, Osamu

2011-01-01

Protein transport across membranes is a fundamental and essential cellular activity in all organisms. In bacteria, protein export across the cytoplasmic membrane, driven by dynamic interplays between the protein-conducting SecYEG channel (Sec translocon) and the SecA ATPase, is enhanced by the proton motive force (PMF) and a membrane-integrated Sec component, SecDF. However, the structure and function of SecDF have remained unclear. We solved the first crystal structure of SecDF, consisting of a pseudo-symmetrical 12-helix transmembrane domain and two protruding periplasmic domains. Based on the structural features, we proposed that SecDF functions as a membrane-integrated chaperone, which drives protein movement without using the major energetic currency, ATP, but with remarkable cycles of conformational changes, powered by the proton gradient across the membrane. By a series of biochemical and biophysical approaches, several functionally important residues in the transmembrane region have been identified and our model of the SecDF function has been verified. PMID:27857601

A mammalian nervous system-specific plasma membrane proteasome complex that modulates neuronal function

PubMed Central

Ramachandran, Kapil V.; Margolis, Seth S.

2017-01-01

In the nervous system, rapidly occurring processes such as neuronal transmission and calcium signaling are affected by short-term inhibition of proteasome function. It remains unclear how proteasomes can acutely regulate such processes, as this is inconsistent with their canonical role in proteostasis. Here, we made the discovery of a mammalian nervous system-specific membrane proteasome complex that directly and rapidly modulates neuronal function by degrading intracellular proteins into extracellular peptides that can stimulate neuronal signaling. This proteasome complex is tightly associated with neuronal plasma membranes, exposed to the extracellular space, and catalytically active. Selective inhibition of this membrane proteasome complex by a cell-impermeable proteasome inhibitor blocked extracellular peptide production and attenuated neuronal activity-induced calcium signaling. Moreover, membrane proteasome-derived peptides are sufficient to induce neuronal calcium signaling. Our discoveries challenge the prevailing notion that proteasomes primarily function to maintain proteostasis, and highlight a form of neuronal communication through a membrane proteasome complex. PMID:28287632

RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

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

Bello, Oscar D.; Zanetti, M. Natalia; Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM

2012-03-10

Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggestedmore » as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: Black-Right-Pointing-Pointer RIM and Munc13 are present in human sperm and localize to the acrosomal region. Black-Right-Pointing-Pointer RIM and Munc13 are necessary for acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Munc13 participate before the acrosomal calcium efflux. Black-Right-Pointing-Pointer RIM, Munc13 and Rab3A interplay in human sperm acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Rab3A have critical roles in membrane docking.« less

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

PubMed Central

Kraft, Mary L.

2017-01-01

Sphingolipids are structural components in the plasma membranes of eukaryotic cells. Their metabolism produces bioactive signaling molecules that modulate fundamental cellular processes. The segregation of sphingolipids into distinct membrane domains is likely essential for cellular function. This review presents the early studies of sphingolipid distribution in the plasma membranes of mammalian cells that shaped the most popular current model of plasma membrane organization. The results of traditional imaging studies of sphingolipid distribution in stimulated and resting cells are described. These data are compared with recent results obtained with advanced imaging techniques, including super-resolution fluorescence detection and high-resolution secondary ion mass spectrometry (SIMS). Emphasis is placed on the new insight into the sphingolipid organization within the plasma membrane that has resulted from the direct imaging of stable isotope-labeled lipids in actual cell membranes with high-resolution SIMS. Super-resolution fluorescence techniques have recently revealed the biophysical behaviors of sphingolipids and the unhindered diffusion of cholesterol analogs in the membranes of living cells are ultimately in contrast to the prevailing hypothetical model of plasma membrane organization. High-resolution SIMS studies also conflicted with the prevailing hypothesis, showing sphingolipids are concentrated in micrometer-scale membrane domains, but cholesterol is evenly distributed within the plasma membrane. Reductions in cellular cholesterol decreased the number of sphingolipid domains in the plasma membrane, whereas disruption of the cytoskeleton eliminated them. In addition, hemagglutinin, a transmembrane protein that is thought to be a putative raft marker, did not cluster within sphingolipid-enriched regions in the plasma membrane. Thus, sphingolipid distribution in the plasma membrane is dependent on the cytoskeleton, but not on favorable interactions with cholesterol or hemagglutinin. The alternate views of plasma membrane organization suggested by these findings are discussed. PMID:28119913

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

PubMed

Kraft, Mary L

2016-01-01

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

PERVAPORATION MEMBRANE SYSTEMS FOR VOLATILE FERMENTATION PRODUCT RECOVERY AND DEHYDRATION

EPA Science Inventory

The economics of fermentative production of fuels and commodity chemicals can be a strong function of the efficiency with which the fermentation products are removed from the biological media. Due to growth inhibition by some fermentation products, including ethanol, concentrati...

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton

PubMed Central

2017-01-01

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria. PMID:28945742

The bacterial Sec system is required for the organization and function of the MreB cytoskeleton.

PubMed

Govindarajan, Sutharsan; Amster-Choder, Orna

2017-09-01

The Sec system is responsible for protein insertion, translocation and secretion across membranes in all cells. The bacterial actin homolog MreB controls various processes, including cell wall synthesis, membrane organization and polarity establishment. Here we show that the two systems genetically interact and that components of the Sec system, especially the SecA motor protein, are essential for spatiotemporal organization of MreB in E. coli, as evidenced by the accumulation of MreB at irregular sites in Sec-impaired cells. MreB mislocalization in SecA-defective cells significantly affects MreB-coordinated processes, such as cell wall synthesis, and induce formation of membrane invaginations enriched in high fluidity domains. Additionally, MreB is not recruited to the FtsZ ring in secA mutant cells, contributing to division arrest and cell filamentation. Our results show that all these faults are due to improper targeting of MreB to the membrane in the absence of SecA. Thus, when we reroute RodZ, MreB membrane-anchor, by fusing it to a SecA-independent integral membrane protein and overproducing it, MreB localization is restored and the defect in cell division is corrected. Notably, the RodZ moiety is not properly inserted into the membrane, strongly suggesting that it only serves as a bait for placing MreB around the cell circumference. Finally, we show that MreB localization depends on SecA also in C. crescentus, suggesting that regulation of MreB by the Sec system is conserved in bacteria. Taken together, our data reveal that the secretion system plays an important role in determining the organization and functioning of the cytoskeletal system in bacteria.

Novel functionalized nano-TiO2 loading electrocatalytic membrane for oily wastewater treatment.

PubMed

Yang, Yang; Wang, Hong; Li, Jianxin; He, Benqiao; Wang, Tonghua; Liao, Shijun

2012-06-19

Membrane fouling is a critical problem in membrane filtration processes for water purification. Electrocatalytic membrane reactor (ECMR) was an effective method to avoid membrane fouling and improve water quality. This study focuses on the preparation and characterization of a novel functionalized nano-TiO(2) loading electrocatalytic membrane for oily wastewater treatment. A TiO(2)/carbon membrane used in the reactor is prepared by coating TiO(2) as an electrocatalyst via a sol-gel process on a conductive microporous carbon membrane. In order to immobilize TiO(2) on the carbon membrane, the carbon membrane is first pretreated with HNO(3) to generate the oxygen-containing functional groups on its surface. X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), high-resolution transmission electron microscopy (HRTEM), and X-ray photoelectron spectroscopy (XPS) analyses are used to evaluate the morphology and microstructure of the membranes. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) measurements are employed to illustrate the eletrochemical activity of the TiO(2)/carbon membrane. The membrane performance is investigated by treating oily wastewater. The oil removal rate increases with a decrease in the liquid hourly space velocity (LHSV) through the ECMR. The COD removal rate was 100% with a LHSV of 7.2 h(-1) and 87.4% with a LHSV of 21.6 h(-1) during the treatment of 200 mg/L oily water. It suggests that the synergistic effect of electrocatalytic oxidation and membrane separation in the ECMR plays a key role.

Protein prenylation: a new mode of host-pathogen interaction.

PubMed

Amaya, Moushimi; Baranova, Ancha; van Hoek, Monique L

2011-12-09

Post translational modifications are required for proteins to be fully functional. The three step process, prenylation, leads to farnesylation or geranylgeranylation, which increase the hydrophobicity of the prenylated protein for efficient anchoring into plasma membranes and/or organellar membranes. Prenylated proteins function in a number of signaling and regulatory pathways that are responsible for basic cell operations. Well characterized prenylated proteins include Ras, Rac and Rho. Recently, pathogenic prokaryotic proteins, such as SifA and AnkB, have been shown to be prenylated by eukaryotic host cell machinery, but their functions remain elusive. The identification of other bacterial proteins undergoing this type of host-directed post-translational modification shows promise in elucidating host-pathogen interactions to develop new therapeutics. This review incorporates new advances in the study of protein prenylation into a broader aspect of biology with a focus on host-pathogen interaction. Copyright © 2011 Elsevier Inc. All rights reserved.

Recombinant G protein-coupled receptor expression in Saccharomyces cerevisiae for protein characterization.

PubMed

Blocker, Kory M; Britton, Zachary T; Naranjo, Andrea N; McNeely, Patrick M; Young, Carissa L; Robinson, Anne S

2015-01-01

G protein-coupled receptors (GPCRs) are membrane proteins that mediate signaling across the cellular membrane and facilitate cellular responses to external stimuli. Due to the critical role that GPCRs play in signal transduction, therapeutics have been developed to influence GPCR function without an extensive understanding of the receptors themselves. Closing this knowledge gap is of paramount importance to improving therapeutic efficacy and specificity, where efforts to achieve this end have focused chiefly on improving our knowledge of the structure-function relationship. The purpose of this chapter is to review methods for the heterologous expression of GPCRs in Saccharomyces cerevisiae, including whole-cell assays that enable quantitation of expression, localization, and function in vivo. In addition, we describe methods for the micellular solubilization of the human adenosine A2a receptor and for reconstitution of the receptor in liposomes that have enabled its biophysical characterization. © 2015 Elsevier Inc. All rights reserved.

Functional profiles of orphan membrane transporters in the life cycle of the malaria parasite

PubMed Central

Kenthirapalan, Sanketha; Waters, Andrew P.; Matuschewski, Kai; Kooij, Taco W. A.

2016-01-01

Assigning function to orphan membrane transport proteins and prioritizing candidates for detailed biochemical characterization remain fundamental challenges and are particularly important for medically relevant pathogens, such as malaria parasites. Here we present a comprehensive genetic analysis of 35 orphan transport proteins of Plasmodium berghei during its life cycle in mice and Anopheles mosquitoes. Six genes, including four candidate aminophospholipid transporters, are refractory to gene deletion, indicative of essential functions. We generate and phenotypically characterize 29 mutant strains with deletions of individual transporter genes. Whereas seven genes appear to be dispensable under the experimental conditions tested, deletion of any of the 22 other genes leads to specific defects in life cycle progression in vivo and/or host transition. Our study provides growing support for a potential link between heavy metal homeostasis and host switching and reveals potential targets for rational design of new intervention strategies against malaria. PMID:26796412

Phytochemicals perturb membranes and promiscuously alter protein function.

PubMed

Ingólfsson, Helgi I; Thakur, Pratima; Herold, Karl F; Hobart, E Ashley; Ramsey, Nicole B; Periole, Xavier; de Jong, Djurre H; Zwama, Martijn; Yilmaz, Duygu; Hall, Katherine; Maretzky, Thorsten; Hemmings, Hugh C; Blobel, Carl; Marrink, Siewert J; Koçer, Armağan; Sack, Jon T; Andersen, Olaf S

2014-08-15

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding.

Phytochemicals Perturb Membranes and Promiscuously Alter Protein Function

PubMed Central

2015-01-01

A wide variety of phytochemicals are consumed for their perceived health benefits. Many of these phytochemicals have been found to alter numerous cell functions, but the mechanisms underlying their biological activity tend to be poorly understood. Phenolic phytochemicals are particularly promiscuous modifiers of membrane protein function, suggesting that some of their actions may be due to a common, membrane bilayer-mediated mechanism. To test whether bilayer perturbation may underlie this diversity of actions, we examined five bioactive phenols reported to have medicinal value: capsaicin from chili peppers, curcumin from turmeric, EGCG from green tea, genistein from soybeans, and resveratrol from grapes. We find that each of these widely consumed phytochemicals alters lipid bilayer properties and the function of diverse membrane proteins. Molecular dynamics simulations show that these phytochemicals modify bilayer properties by localizing to the bilayer/solution interface. Bilayer-modifying propensity was verified using a gramicidin-based assay, and indiscriminate modulation of membrane protein function was demonstrated using four proteins: membrane-anchored metalloproteases, mechanosensitive ion channels, and voltage-dependent potassium and sodium channels. Each protein exhibited similar responses to multiple phytochemicals, consistent with a common, bilayer-mediated mechanism. Our results suggest that many effects of amphiphilic phytochemicals are due to cell membrane perturbations, rather than specific protein binding. PMID:24901212

Structure of the Ebola virus envelope protein MPER/TM domain and its interaction with the fusion loop explains their fusion activity.

PubMed

Lee, Jinwoo; Nyenhuis, David A; Nelson, Elizabeth A; Cafiso, David S; White, Judith M; Tamm, Lukas K

2017-09-19

Ebolavirus (EBOV), an enveloped filamentous RNA virus causing severe hemorrhagic fever, enters cells by macropinocytosis and membrane fusion in a late endosomal compartment. Fusion is mediated by the EBOV envelope glycoprotein GP, which consists of subunits GP1 and GP2. GP1 binds to cellular receptors, including Niemann-Pick C1 (NPC1) protein, and GP2 is responsible for low pH-induced membrane fusion. Proteolytic cleavage and NPC1 binding at endosomal pH lead to conformational rearrangements of GP2 that include exposing the hydrophobic fusion loop (FL) for insertion into the cellular target membrane and forming a six-helix bundle structure. Although major portions of the GP2 structure have been solved in pre- and postfusion states and although current models place the transmembrane (TM) and FL domains of GP2 in close proximity at critical steps of membrane fusion, their structures in membrane environments, and especially interactions between them, have not yet been characterized. Here, we present the structure of the membrane proximal external region (MPER) connected to the TM domain: i.e., the missing parts of the EBOV GP2 structure. The structure, solved by solution NMR and EPR spectroscopy in membrane-mimetic environments, consists of a helix-turn-helix architecture that is independent of pH. Moreover, the MPER region is shown to interact in the membrane interface with the previously determined structure of the EBOV FL through several critical aromatic residues. Mutation of aromatic and neighboring residues in both binding partners decreases fusion and viral entry, highlighting the functional importance of the MPER/TM-FL interaction in EBOV entry and fusion.

Feedback Interactions of Polymerized Actin with the Cell Membrane: Waves, Pulses, and Oscillations

NASA Astrophysics Data System (ADS)

Carlsson, Anders

Polymerized filaments of the protein actin have crucial functions in cell migration, and in bending the cell membrane to drive endocytosis or the formation of protrusions. The nucleation and polymerization of actin filaments are controlled by upstream agents in the cell membrane, including nucleation-promoting factors (NPFs) that activate the Arp2/3 complex to form new branches on pre-existing filaments. But polymerized actin (F-actin) also feeds back on the assembly of NPFs. We explore the effects of the resulting feedback loop of F-actin and NPFs on two phenomena: actin pulses that drive endocytosis in yeast, and actin waves traveling along the membrane of several cell types. In our model of endocytosis in yeast, the actin network is grown explicitly in three dimensions, exerts a negative feedback interaction on localized patch of NPFs in the membrane, and bends the membrane by exerting a distribution of forces. This model explains observed actin and NPF pulse dynamics, and the effects of several interventions including i) NPF mutations, ii) inhibition of actin polymerization, and iii) deletion of a protein that allows F-actin to bend the cell membrane. The model predicts that mutation of the active region of an NPF will enhance the accumulation of that NPF, and we confirm this prediction by quantitative fluorescence microscopy. For actin waves, we treat a similar model, with NPFs distributed over a larger region of the cell membrane. This model naturally generates actin waves, and predicts a transition from wave behavior to spatially localized oscillations when NPFs are confined to a small region. We also predict a transition from waves to static polarization as the negative-feedback coupling between F-actin and the NPFs is reduced. Supported by NIGMS Grant R01 GM107667.

Diversity and regulation of plant Ca2+ pumps: insights from expression in yeast

NASA Technical Reports Server (NTRS)

Sze, H.; Liang, F.; Hwang, I.; Curran, A. C.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

The spatial and temporal regulation of calcium concentration in plant cells depends on the coordinate activities of channels and active transporters located on different organelles and membranes. Several Ca2+ pumps have been identified and characterized by functional expression of plant genes in a yeast mutant (K616). This expression system has opened the way to a genetic and biochemical characterization of the regulatory and catalytic features of diverse Ca2+ pumps. Plant Ca(2+)-ATPases fall into two major types: AtECA1 represents one of four or more members of the type IIA (ER-type) Ca(2+)-ATPases in Arabidopsis, and AtACA2 is one of seven or more members of the type IIB (PM-type) Ca(2+)-ATPases that are regulated by a novel amino terminal domain. Type IIB pumps are widely distributed on membranes, including the PM (plasma membrane), vacuole, and ER (endoplasmic reticulum). The regulatory domain serves multiple functions, including autoinhibition, calmodulin binding, and sites for modification by phosphorylation. This domain, however, is considerably diverse among several type IIB ATPases, suggesting that the pumps are differentially regulated. Understanding of Ca2+ transporters at the molecular level is providing insights into their roles in signaling networks and in regulating fundamental processes of cell biology.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

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

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylationmore » target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.« less

Self-assembled tethered bimolecular lipid membranes.

PubMed

Sinner, Eva-Kathrin; Ritz, Sandra; Naumann, Renate; Schiller, Stefan; Knoll, Wolfgang

2009-01-01

This chapter describes some of the strategies developed in our group for designing, constructing and structurally and functionally characterizing tethered bimolecular lipid membranes (tBLM). We introduce this platform as a novel model membrane system that complements the existing ones, for example, Langmuir monolayers, vesicular liposomal dispersions and bimolecular ("black") lipid membranes. Moreover, it offers the additional advantage of allowing for studies of the influence of membrane structure and order on the function of integral proteins, for example, on how the composition and organization of lipids in a mixed membrane influence the ion translocation activity of integral channel proteins. The first strategy that we introduce concerns the preparation of tethered monolayers by the self-assembly of telechelics. Their molecular architecture with a headgroup, a spacer unit (the "tether") and the amphiphile that mimics the lipid molecule allows them to bind specifically to the solid support thus forming the proximal layer of the final architecture. After fusion of vesicles that could contain reconstituted proteins from a liposomal dispersion in contact to this monolayer the tethered bimolecular lipid membrane is obtained. This can then be characterized by a broad range of surface analytical techniques, including surface plasmon spectroscopies, the quartz crystal microbalance, fluorescence and IR spectroscopies, and electrochemical techniques, to mention a few. It is shown that this concept allows for the construction of tethered lipid bilayers with outstanding electrical properties including resistivities in excess of 10 MOmega cm2. A modified strategy uses the assembly of peptides as spacers that couple covalently via their engineered sulfhydryl or lipoic acid groups at the N-terminus to the employed gold substrate, while their C-terminus is being activated afterward for the coupling of, for example, dimyristoylphosphatidylethanol amine (DMPE) lipid molecules via the NH2 moiety of their headgroups. It is demonstrated that these membranes are well suited for the in situ synthesis of membrane protein by a cell-free expression approach. The vectorial integration of an in vitro synthesized odorant receptor, OR5 from the rat, is demonstrated by means of antibodies that specifically bind to a tag at the N-terminus of the receptor and is read out by surface plasmon fluorescence spectroscopy. A completely different strategy employs his-tagged membrane proteins in their solubilized form binding to a surface-attached Ni(+)-NTA monolayer generating a well-oriented protein layer the density of which can be easily controlled by online monitoring the binding (assembly) step by surface plasmon spectroscopy. Moreover, the attachment of the his-tag to either the C- or the N-terminus allows for the complete control of the protein orientation. After the exchange of the detergent micelle by a lipid bilayer via a surface dialysis procedure an electrically very well isolating protein-tethered membrane is formed. We show that this "wiring" of the functional units allows for the (external) manipulation of the oxidation state of the redox-protein cytochrome c Oxidase by the control of the potential applied to the gold substrate which is used as the working electrode in an electrochemical attachment.

The urothelium: anatomy, review of the literature, perspectives for veterinary medicine.

PubMed

Arrighi, S

2015-03-01

Over time, much knowledge has been accumulated about the active role of the urothelium, principally in rodents and human. Far from being a mere passive barrier, this specialized epithelium can alter the ion and protein composition of the urine, is able to sense and respond to mechanical stimuli such as pressure, and react to mechanical stimuli by epithelial cell communication with the nervous system. Most of the specialized functions of the urothelium are linked to a number of morpho-physiologic properties exhibited by the superficial umbrella cells, including specialized membrane lipids, asymmetric unit membrane particles and a plasmalemma with stiff plaques which function as a barrier to most substances found in urine, thus protecting the underlying tissues. Moreover, the entire mucosa lining the low urinary tract, composed of urothelium and sub-urothelium, forms a functional transduction unit, able to respond to eso- and endogenous physical and chemical stimuli in a manner assuring an adequate functional response. This review will summarize the available information on each area of inquiry from a morpho-functional point of view. Possible considerations pertaining to species of veterinary interest are reviewed as well. The review was prepared consulting the electronic databases PubMed and Cab Abstracts and retrieving all pertinent reports and the relative reference lists, in order to identify any potential additional studies that could be included. Full-length research articles and thematic reviews were considered. Information on the urothelium of some domestic animal species was also included. Copyright © 2014 Elsevier GmbH. All rights reserved.

Calcium accelerates SNARE-mediated lipid mixing through modulating α-synuclein membrane interaction.

PubMed

Zhang, Zeting; Jiang, Xin; Xu, Danrui; Zheng, Wenwen; Liu, Maili; Li, Conggang

2018-04-04

α-Synuclein is involved in Parkinson's disease, and its interaction with cell membrane is vital to its pathological and physiological functions. We have shown that Ca 2+ can regulate α-synuclein membrane interaction, but the physiological role of Ca 2+ in modulating α-synuclein membrane interaction is still unexplored. Based on the previous findings that α-synuclein inhibits membrane fusion and its inhibitory effect is highly related to its membrane binding, here we employed solution state Nuclear Magnetic Resonance (NMR) spectroscopy and the ensemble fluorescence fusion assay to show that Ca 2+ can modulate the inhibitory effect of α-synuclein on SNARE-mediated membrane fusion through disrupting α-synuclein membrane interaction, resulting in acceleration of SNARE-mediated membrane fusion. These results suggest a modulatory effect of Ca 2+ on membrane mediated normal function of α-synuclein, which of importance for the study of the Parkinson's disease. Copyright © 2018 Elsevier B.V. All rights reserved.

Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling

PubMed Central

Diaz, Maira; Sanchez-Barrena, Maria Jose; Gonzalez-Rubio, Juana Maria; Rodriguez, Lesia; Fernandez, Daniel; Antoni, Regina; Yunta, Cristina; Belda-Palazon, Borja; Gonzalez-Guzman, Miguel; Peirats-Llobet, Marta; Menendez, Margarita; Boskovic, Jasminka; Marquez, Jose A.; Rodriguez, Pedro L.; Albert, Armando

2016-01-01

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca2+ are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca2+ signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca2+-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca2+ sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca2+-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress. PMID:26719420

Calcium-dependent oligomerization of CAR proteins at cell membrane modulates ABA signaling.

PubMed

Diaz, Maira; Sanchez-Barrena, Maria Jose; Gonzalez-Rubio, Juana Maria; Rodriguez, Lesia; Fernandez, Daniel; Antoni, Regina; Yunta, Cristina; Belda-Palazon, Borja; Gonzalez-Guzman, Miguel; Peirats-Llobet, Marta; Menendez, Margarita; Boskovic, Jasminka; Marquez, Jose A; Rodriguez, Pedro L; Albert, Armando

2016-01-19

Regulation of ion transport in plants is essential for cell function. Abiotic stress unbalances cell ion homeostasis, and plants tend to readjust it, regulating membrane transporters and channels. The plant hormone abscisic acid (ABA) and the second messenger Ca(2+) are central in such processes, as they are involved in the regulation of protein kinases and phosphatases that control ion transport activity in response to environmental stimuli. The identification and characterization of the molecular mechanisms underlying the effect of ABA and Ca(2+) signaling pathways on membrane function are central and could provide opportunities for crop improvement. The C2-domain ABA-related (CAR) family of small proteins is involved in the Ca(2+)-dependent recruitment of the pyrabactin resistance 1/PYR1-like (PYR/PYL) ABA receptors to the membrane. However, to fully understand CAR function, it is necessary to define a molecular mechanism that integrates Ca(2+) sensing, membrane interaction, and the recognition of the PYR/PYL interacting partners. We present structural and biochemical data showing that CARs are peripheral membrane proteins that functionally cluster on the membrane and generate strong positive membrane curvature in a Ca(2+)-dependent manner. These features represent a mechanism for the generation, stabilization, and/or specific recognition of membrane discontinuities. Such structures may act as signaling platforms involved in the recruitment of PYR/PYL receptors and other signaling components involved in cell responses to stress.

A role for protein kinase C in the regulation of membrane fluidity and Ca²(+) flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells.

PubMed

Chen, Lihong; Meng, Qingli; Jing, Xian; Xu, Pingxiang; Luo, Dali

2011-02-01

Protein kinase C (PKC) plays a prominent role in the regulation of a variety of cellular functions, including Ca²(+) signalling. In HEK293 and Jurkat cells, the Ca²(+) release and Ca²(+) uptake stimulated by several different activators were attenuated by activation of PKC with phorbol myristate acetate (PMA) or 1-oleoyl-2-acetyl-sn-glycerol (OAG) and potentiated by PKC inhibition with Gö6983 or knockdown of PKCα or PKCβ using shRNA. Immunostaining and Western blotting analyses revealed that PKCα and PKCβII accumulated at the plasma membrane (PM) and that these isoforms, along with PKCβI, also translocated to the endoplasmic reticulum (ER) upon activation with PMA. Measurements of membrane fluidity showed that, like the cell membrane stabilizers bovine serum albumin (BSA) and ursodeoxycholate (UDCA), PMA and OAG significantly reduced the fluidity of both the PM and ER membranes; these effects were blocked in PKC-knockdown cells. Interestingly, both BSA and UDCA inhibited the Ca²(+) responses to agonists to the same extent as PMA, whereas Tween 20, which increases membrane fluidity, raised the internal Ca²(+) concentration. Thus, activation of PKC induces both translocation of PKC to the PM and ER membranes and downregulation of membrane fluidity, thereby negatively modulating Ca²(+) flux. Copyright © 2010 Elsevier Inc. All rights reserved.

Membrane Perturbation Induced by Interfacially Adsorbed Peptides

PubMed Central

Zemel, Assaf; Ben-Shaul, Avinoam; May, Sylvio

2004-01-01

The structural and energetic characteristics of the interaction between interfacially adsorbed (partially inserted) α-helical, amphipathic peptides and the lipid bilayer substrate are studied using a molecular level theory of lipid chain packing in membranes. The peptides are modeled as “amphipathic cylinders” characterized by a well-defined polar angle. Assuming two-dimensional nematic order of the adsorbed peptides, the membrane perturbation free energy is evaluated using a cell-like model; the peptide axes are parallel to the membrane plane. The elastic and interfacial contributions to the perturbation free energy of the “peptide-dressed” membrane are evaluated as a function of: the peptide penetration depth into the bilayer's hydrophobic core, the membrane thickness, the polar angle, and the lipid/peptide ratio. The structural properties calculated include the shape and extent of the distorted (stretched and bent) lipid chains surrounding the adsorbed peptide, and their orientational (C-H) bond order parameter profiles. The changes in bond order parameters attendant upon peptide adsorption are in good agreement with magnetic resonance measurements. Also consistent with experiment, our model predicts that peptide adsorption results in membrane thinning. Our calculations reveal pronounced, membrane-mediated, attractive interactions between the adsorbed peptides, suggesting a possible mechanism for lateral aggregation of membrane-bound peptides. As a special case of interest, we have also investigated completely hydrophobic peptides, for which we find a strong energetic preference for the transmembrane (inserted) orientation over the horizontal (adsorbed) orientation. PMID:15189858

Peroxisomal membrane ascorbate peroxidase is sorted to a membranous network that resembles a subdomain of the endoplasmic reticulum.

PubMed Central

Mullen, R T; Lisenbee, C S; Miernyk, J A; Trelease, R N

1999-01-01

The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD(+) and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3'-dihexyloxacarbocyanine iodide-stained membranes) and to peroxisomes. The reticular network did not colocalize with other organelle marker proteins, including three ER reticuloplasmins. However, in vitro, peroxisomal APX inserted post-translationally into the ER but not into other purified organelle membranes (including peroxisomal membranes). Insertion into the ER depended on the presence of molecular chaperones and ATP. These results suggest that regions of the ER serve as a possible intermediate in the sorting pathway of peroxisomal APX. Insight into this hypothesis was obtained from in vivo experiments with brefeldin A (BFA), a toxin that blocks vesicle-mediated protein export from ER. A transiently expressed chloramphenicol acetyltransferase-peroxisomal APX (CAT-pAPX) fusion protein accumulated only in the reticular/circular network in BFA-treated cells; after subsequent removal of BFA from these cells, the CAT-pAPX was distributed to preexisting peroxisomes. Thus, plant peroxisomal APX, a representative enzymatic peroxisomal membrane protein, is sorted to peroxisomes through an indirect pathway involving a preperoxisomal compartment with characteristics of a distinct subdomain of the ER, possibly a peroxisomal ER subdomain. PMID:10559442

Membrane proteins bind lipids selectively to modulate their structure and function.

PubMed

Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V

2014-06-05

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding.

The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

PubMed

Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

2015-01-01

The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles*S⃞

PubMed Central

Cappuccio, Jenny A.; Blanchette, Craig D.; Sulchek, Todd A.; Arroyo, Erin S.; Kralj, Joel M.; Hinz, Angela K.; Kuhn, Edward A.; Chromy, Brett A.; Segelke, Brent W.; Rothschild, Kenneth J.; Fletcher, Julia E.; Katzen, Federico; Peterson, Todd C.; Kudlicki, Wieslaw A.; Bench, Graham; Hoeprich, Paul D.; Coleman, Matthew A.

2008-01-01

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies. PMID:18603642

Plant Ion Channels: Gene Families, Physiology, and Functional Genomics Analyses

PubMed Central

Ward, John M.; Mäser, Pascal; Schroeder, Julian I.

2016-01-01

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization-and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide–gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport. PMID:18842100

Plant ion channels: gene families, physiology, and functional genomics analyses.

PubMed

Ward, John M; Mäser, Pascal; Schroeder, Julian I

2009-01-01

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

A microfabricated platform with hydrogel arrays for 3D mechanical stimulation of cells.

PubMed

Liu, Haijiao; Usprech, Jenna; Sun, Yu; Simmons, Craig A

2016-04-01

Cellular microenvironments present cells with multiple stimuli, including not only soluble biochemical and insoluble matrix cues but also mechanical factors. Biomaterial array platforms have been used to combinatorially and efficiently probe and define two-dimensional (2D) and 3D microenvironmental cues to guide cell functions for tissue engineering applications. However, there are few examples of array platforms that include dynamic mechanical forces, particularly to enable stretching of 3D cell-seeded biomaterials, which is relevant to engineering connective and cardiovascular tissues. Here we present a deformable membrane platform that enables 3D dynamic mechanical stretch of arrayed biomaterial constructs. Cell-seeded polyethylene glycol norbornene (PEG-NB) hydrogels were bound to miniaturized deformable membranes via a thiol-ene reaction with off-stoichiometry thiol-ene based polydimethylsiloxane (OSTE-PDMS) as the membrane material. Bonding to OSTE-PDMS enabled the 3D hydrogel microconstructs to be cyclically deformed and stretched by the membrane. As a first demonstration, human mesenchymal stromal cells (MSCs) embedded in PEG-NB were stretched for several days. They were found to be viable, spread in the 3D hydrogels, and exhibited a contractile myofibroblast phenotype when exposed to dynamic 3D mechanical deformation. This platform, which is readily scalable to larger arrays, enables systematic interrogation of the relationships between combinations of 3D mechanobiological cues and cellular responses, and thus has the potential to identify strategies to predictably control the construction of functional engineered tissues. Current high-throughput biomaterial screening approaches fail to consider the effects of dynamic mechanical stimulation, despite its importance in a wide variety of regenerative medicine applications. To meet this need, we developed a deformable membrane platform that enables 3D dynamic stretch of arrayed biomaterial constructs. Our approach combines microtechnologies fabricated with off-stoichiometry thiol-ene based polydimethylsiloxane membranes that can covalently bond cell-seeded polyethylene glycol norbornene 3D hydrogels, a model biomaterial with tunable adhesive, elastic and degradation characteristics. As a first demonstration, we show that human mesenchymal stromal cells embedded in hydrogels and subjected to dynamic mechanical stimulation undergo myofibroblast differentiation. This system is readily scaled up to larger arrays, and will enable systematic and efficient screening of combinations of 3D mechanobiological and biomaterial cues on cell fate and function. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Multilayer Membranes of Glycosaminoglycans and Collagen I Biomaterials Modulate the Function and Microvesicle Release of Endothelial Progenitor Cells.

PubMed

Dai, Bingyan; Pan, Qunwen; Li, Zhanghua; Zhao, Mingyan; Liao, Xiaorong; Wu, Keng; Ma, Xiaotang

2016-01-01

Multilayer composite membrane of biomaterials can increase the function of adipose stem cells or osteoprogenitor cells. Recent evidence indicates endothelial progenitor cells (EPCs) and EPCs released microvesicles (MVs) play important roles in angiogenesis and vascular repair. Here, we investigated the effects of biomaterial multilayer membranes of hyaluronic acid (HA) or chondroitin sulfate (CS) and Collagen I (Col I) on the functions and MVs release of EPCs. Layer-by-layer (LBL) technology was applied to construct the multilayer composite membranes. Four types of the membranes constructed by adsorbing either HA or CS and Col I alternatively with different top layers were studied. The results showed that all four types of multilayer composite membranes could promote EPCs proliferation and migration and inhibit cell senility, apoptosis, and the expression of activated caspase-3. Interestingly, these biomaterials increased the release and the miR-126 level of EPCs-MVs. Moreover, the CS-Col I membrane with CS on the top layer showed the most effects on promoting EPCs proliferation, EPCs-MV release, and miR-126 level in EPCs-MVs. In conclusion, HA/CS and Collagen I composed multilayer composite membranes can promote EPCs functions and release of miR-126 riched EPCs-MVs, which provides a novel strategy for tissue repair treatment.

Improving the performance of water desalination through ultra-permeable functionalized nanoporous graphene oxide membrane

NASA Astrophysics Data System (ADS)

Hosseini, Mostafa; Azamat, Jafar; Erfan-Niya, Hamid

2018-01-01

Molecular dynamics simulations were performed to investigate the water desalination performance of nanoporous graphene oxide (NPGO) membranes. The simulated systems consist of a NPGO as a membrane with a functionalized pore in its center immersed in an aqueous ionic solution and a graphene sheet as a barrier. The considered NPGO membranes are involved four types of pore with different size and chemistry. The results indicated that the NPGO membrane has effective efficiency in salt rejection as well as high performance in water flux. For all types of pore with the radius size of 2.9-4.5 Å, the NPGO shows salt rejection of >89%. Functional groups on the surface and edge of pores have a great effect on water flux. To precisely understand the effect of functional groups on the surface of nanostructured membranes, nanoporous graphene was simulated under the same condition for comparison. Hydrophilic groups on the surface make the NPGO as an ultra-permeable membrane. As a result, the obtained water flux for NPGO was about 77% greater than graphene. Also, it was found that the water flux of NPGO is 2-5 orders of magnitude greater than other existing reverse osmosis membranes. Therefore, the investigated systems can be recommended as a model for the water desalination.

CO2 Acquisition Membrane (CAM)

NASA Technical Reports Server (NTRS)

Mason, Larry W.; Way, J. Douglas; Vlasse, Marcus

2003-01-01

The objective of CAM is to develop, test, and analyze thin film membrane materials for separation and purification of carbon dioxide (CO2) from mixtures of gases, such as those found in the Martian atmosphere. The membranes are targeted toward In Situ Resource Utilization (ISRU) applications that will operate in extraterrestrial environments and support future unmanned and human space missions. A primary application is the Sabatier Electrolysis process that uses Mars atmosphere CO2 as raw material for producing water, oxygen, and methane for rocket fuel and habitat support. Other applications include use as an inlet filter to collect and concentrate Mars atmospheric argon and nitrogen gases for habitat pressurization, and to remove CO2 from breathing gases in Closed Environment Life Support Systems (CELSS). CAM membrane materials include crystalline faujasite (FAU) zeolite and rubbery polymers such as silicone rubber (PDMS) that have been shown in the literature and via molecular simulation to favor adsorption and permeation of CO2 over nitrogen and argon. Pure gas permeation tests using commercial PDMS membranes have shown that both CO2 permeance and the separation factor relative to other gases increase as the temperature decreases, and low (Delta)P(Sub CO2) favors higher separation factors. The ideal CO2/N2 separation factor increases from 7.5 to 17.5 as temperature decreases from 22 C to -30 C. For gas mixtures containing CO2, N2, and Ar, plasticization decreased the separation factors from 4.5 to 6 over the same temperature range. We currently synthesize and test our own Na(+) FAU zeolite membranes using standard formulations and secondary growth methods on porous alumina. Preliminary tests with a Na(+) FAU membrane at 22 C show a He/SF6 ideal separation factor of 62, exceeding the Knudsen diffusion selectivity by an order of magnitude. This shows that the membrane is relatively free from large defects and associated non-selective (viscous flow) transport mechanisms. The Membrane Test Facility (MTF) has been developed to measure membrane permeance over a wide range of temperature and pressure. The facility uses two volume compartments separated by the membrane that are instrumented to measure temperature, delta pressure across the membrane, and gas composition. A thermal shroud supports and encloses the membrane, and provides temperature control. Methods were developed to determine membrane permeance using the first order decay of the pressure difference between the sealed compartments, using the total pressure for pure gases, and partial pressure of each species in gas mixtures. The technique provides an end-to-end measurement of gas permeance that includes concentration polarization effects. Experiments have shown that in addition to membrane permeance properties, the geometry and design of associated structures play an important role in how membrane systems will function on Mars.

Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40*

PubMed Central

Kuszak, Adam J.; Jacobs, Daniel; Gurnev, Philip A.; Shiota, Takuya; Louis, John M.; Lithgow, Trevor; Bezrukov, Sergey M.; Rostovtseva, Tatiana K.; Buchanan, Susan K.

2015-01-01

Nearly all mitochondrial proteins are coded by the nuclear genome and must be transported into mitochondria by the translocase of the outer membrane complex. Tom40 is the central subunit of the translocase complex and forms a pore in the mitochondrial outer membrane. To date, the mechanism it utilizes for protein transport remains unclear. Tom40 is predicted to comprise a membrane-spanning β-barrel domain with conserved α-helical domains at both the N and C termini. To investigate Tom40 function, including the role of the N- and C-terminal domains, recombinant forms of the Tom40 protein from the yeast Candida glabrata, and truncated constructs lacking the N- and/or C-terminal domains, were functionally characterized in planar lipid membranes. Our results demonstrate that each of these Tom40 constructs exhibits at least four distinct conductive levels and that full-length and truncated Tom40 constructs specifically interact with a presequence peptide in a concentration- and voltage-dependent manner. Therefore, neither the first 51 amino acids of the N terminus nor the last 13 amino acids of the C terminus are required for Tom40 channel formation or for the interaction with a presequence peptide. Unexpectedly, substrate binding affinity was dependent upon the Tom40 state corresponding to a particular conductive level. A model where two Tom40 pores act in concert as a dimeric protein complex best accounts for the observed biochemical and electrophysiological data. These results provide the first evidence for structurally distinct Tom40 conformations playing a role in substrate recognition and therefore in transport function. PMID:26336107

Magnetic capture of polydopamine-encapsulated Hela cells for the analysis of cell surface proteins.

PubMed

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

2018-02-10

A novel method to characterize cell surface proteins and complexes has been developed. Polydopamine (PDA)-encapsulated Hela cells were prepared for plasma membrane proteome research. Since the PDA protection, the encapsulated cells could be maintained for more than two weeks. Amino groups functionalized magnetic nanoparticles were also used for cell capture by the reaction with the PDA coatings. Plasma membrane fragments were isolated and enriched with assistance of an external magnetic field after disruption of the coated cells by ultrasonic treatment. Plasma membrane proteins (PMPs) and complexes were well preserved on the fragments and identified by shot-gun proteomic analytical strategy. 385 PMPs and 1411 non-PMPs were identified using the method. 85.2% of these PMPs were lipid-raft associated proteins. Ingenuity Pathway Analysis was employed for bio-information extraction from the identified proteins. It was found that 653 non-PMPs had interactions with 140 PMPs. Among them, epidermal growth factor receptor and its complexes, and a series of important pathways including STAT3 pathway were observed. All these results demonstrated that the new approach is of great importance in applying to the research of physiological function and mechanism of the plasma membrane proteins. This work developed a novel strategy for the proteomic analysis of cell surface proteins. According to the results, 73.3% of total identified proteins were lipid-raft associated proteins, which imply that the proposed method is of great potential in the identification of lipid-raft associated proteins. In addition, a series of protein-protein interactions and pathways related to Hela cells were pointed out. All these results demonstrated that our proposed approach is of great importance and could well be applied to the physiological function and mechanism research of plasma membrane proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

When is a cell not a cell? A theory relating coenocytic structure to the unusual electrophysiology of Ventricaria ventricosa (Valonia ventricosa).

PubMed

Shepherd, V A; Beilby, M J; Bisson, M A

2004-06-01

Ventricaria ventricosa and its relatives have intrigued cell biologists and electrophysiologists for over a hundred years. Historically, electrophysiologists have regarded V. ventricosa as a large single plant cell with unusual characteristics including a small and positive vacuole-to-outside membrane potential difference. However, V. ventricosa has a coenocytic construction, with an alveolate cytoplasm interpenetrated by a complex vacuole containing sulphated polysaccharides. We present a theory relating the coenocytic structure to the unusual electrophysiology of V. ventricosa. The alveolate cytoplasm of V. ventricosa consists of a collective of uninucleate cytoplasmic domains interconnected by fine cytoplasmic strands containing microtubules. The cytoplasm is capable of disassociating into single cytoplasmic domains or aggregations of domains that can regenerate new coenocytes. The cytoplasmic domains are enclosed by outer (apical) and inner (basolateral) faces of a communal membrane with polarised K(+)-transporting functions, stabilised by microtubules and resembling a tissue such as a polarised epithelium. There is evidence for membrane trafficking through endocytosis and exocytosis and so "plasmalemma" and "tonoplast" do not have fixed identities. Intra- and extracellular polysaccharide mucilage has effects on electrophysiology through reducing the activity of water and through ion exchange. The vacuole-to-outside potential difference, at which the cell membrane conductance is maximal, reverses its sign from positive under hypertonic conditions to negative under hypotonic conditions. The marked mirror symmetry of the characteristics of current as a function of voltage and conductance as a function of voltage is interpreted as a feature of the communal membrane with polarised K(+) transport. The complex inhomogeneous structure of the cytoplasm places in doubt previous measurements of cytoplasm-to-outside potential difference.

Functional and Morphological Correlations before and after Video-Documented 23-Gauge Pars Plana Vitrectomy with Membrane and ILM Peeling in Patients with Macular Pucker.

PubMed

Mayer, Wolfgang J; Fazekas, Clara; Schumann, Ricarda; Wolf, Armin; Compera, Denise; Kampik, Anselm; Haritoglou, Christos

2015-01-01

Purpose. To assess functional and morphological alterations following video-documented surgery for epiretinal membranes. Methods. Forty-two patients underwent video-documented 23-gauge vitrectomy with peeling of epiretinal (ERM) and inner limiting membrane (ILM). Patient assessment was performed before and 3 and 6 months including best corrected visual acuity (BCVA), slit lamp biomicroscopy, SD-OCT, and central 2° and 18° microperimetry. In addition, all video-documented areas of peeling on the retinal surface were evaluated postoperatively using an additional focal 2° microperimetry. Retinal sensitivity and BCVA were correlated with morphological changes (EZ and ELM) in the foveal region and in regions of membrane peeling. Results. Overall, BCVA increased from 0.6 (±0.2) to 0.2 (±0.2) logMAR after 6 months with an increase in retinal sensitivity (17.9 ± 2.7 dB to 26.8 ± 3.1 dB, p < 0.01). We observed a significant correlation between the integrity of the EZ but not of the ELM and the retinal sensitivity, overall and in peeling areas (p < 0.05). However, no significant correlation between alterations in the area of peeling and overall retinal sensitivity regarding visual acuity gain could be observed after 6 months (p > 0.05). In contrast, overall postoperative retinal sensitivity was significantly decreased in patients with a visual acuity gain lower than 2 lines (p < 0.05) correlating with EZ defects seen in OCT. Conclusions. Mechanical trauma of epiretinal membrane and ILM peeling due to the use of intraocular forceps may affect the outer retinal structure. Nevertheless, these changes seem to have no significant impact on postoperative functional outcome.

Photodynamic Modification Of Plasma Membrane Function In Erythrocytes Sensitized By Xanthenes: What Determines Potency?

NASA Astrophysics Data System (ADS)

Pooler, John P.

1988-02-01

Several xanthene sensitizers were compared as sensitizers of membrane function in erythrocytes and some of their physico-chemical properties were examined. Eosin derivatives that localize at different membrane sites were equally effective at sensitizing both ion leaks and inactivation of membrane cholinesterase, implying that a diffusible intermediate reacts with membrane targets. Assessments of membrane loading and calculations of diffusion distances for singlet oxygen indicate that amounts of membrane-located sensitizer are quantitatively much greater than amounts in the nearby reaction medium. Potency measurements and assessment of absorption spectra and singlet oxygen production in water-dioxane mixtures lead to the conclusion that differential sorption to membranes, photon capture in low polarity environments and conversion of excited states to singlet oxygen are they key determinants of sensitizing potency.

Membrane curvature and its generation by BAR proteins

PubMed Central

Mim, Carsten; Unger, Vinzenz M

2012-01-01

Membranes are flexible barriers that surround the cell and its compartments. To execute vital functions such as locomotion or receptor turnover, cells need to control the shapes of their membranes. In part, this control is achieved through membrane-bending proteins, such as the bin/amphiphysin/rvs domain (BAR) proteins. Many open questions remain about the mechanisms by which membrane-bending proteins function. Addressing this shortfall, recent structures of BAR protein:membrane complexes support existing mechanistic models, but also produced novel insights into how BAR-domain proteins sense, stabilize and generate curvature. Here we review these recent findings, focusing on how BAR proteins interact with the membrane, and how the resulting scaffold structures might aid the recruitment of other proteins to the sites where membranes are bent. PMID:23058040

Composite S-layer lipid structures

PubMed Central

Schuster, Bernhard; Sleytr, Uwe B.

2010-01-01

Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state of the art survey how S-layer proteins, lipids, and polysaccharides may be used as basic building blocks for the assembly of S-layer supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas for application of composite S-layer membrane systems concern sensor systems involving specific membrane functions. PMID:19303933

Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4;#8201;Å

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

Johnson, Zachary Lee; Cheong, Cheom-Gil; Lee, Seok-Yong

2012-07-11

Nucleosides are required for DNA and RNA synthesis, and the nucleoside adenosine has a function in a variety of signalling processes. Transport of nucleosides across cell membranes provides the major source of nucleosides in many cell types and is also responsible for the termination of adenosine signalling. As a result of their hydrophilic nature, nucleosides require a specialized class of integral membrane proteins, known as nucleoside transporters (NTs), for specific transport across cell membranes. In addition to nucleosides, NTs are important determinants for the transport of nucleoside-derived drugs across cell membranes. A wide range of nucleoside-derived drugs, including anticancer drugsmore » (such as Ara-C and gemcitabine) and antiviral drugs (such as zidovudine and ribavirin), have been shown to depend, at least in part, on NTs for transport across cell membranes. Concentrative nucleoside transporters, members of the solute carrier transporter superfamily SLC28, use an ion gradient in the active transport of both nucleosides and nucleoside-derived drugs against their chemical gradients. The structural basis for selective ion-coupled nucleoside transport by concentrative nucleoside transporters is unknown. Here we present the crystal structure of a concentrative nucleoside transporter from Vibrio cholerae in complex with uridine at 2.4 {angstrom}. Our functional data show that, like its human orthologues, the transporter uses a sodium-ion gradient for nucleoside transport. The structure reveals the overall architecture of this class of transporter, unravels the molecular determinants for nucleoside and sodium binding, and provides a framework for understanding the mechanism of nucleoside and nucleoside drug transport across cell membranes.« less

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

PubMed Central

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

2014-01-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham - Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies. PMID:25484487

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

PubMed

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

2014-10-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across the various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham - Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies.

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

NASA Astrophysics Data System (ADS)

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

2014-10-01

Biological membranes constitute boundaries of cells and cell organelles. These membranes are soft fluid interfaces whose thermodynamic states are dictated by bending moduli, induced curvature fields, and thermal fluctuations. Recently, there has been a flood of experimental evidence highlighting active roles for these structures in many cellular processes ranging from trafficking of cargo to cell motility. It is believed that the local membrane curvature, which is continuously altered due to its interactions with myriad proteins and other macromolecules attached to its surface, holds the key to the emergent functionality in these cellular processes. Mechanisms at the atomic scale are dictated by protein-lipid interaction strength, lipid composition, lipid distribution in the vicinity of the protein, shape and amino acid composition of the protein, and its amino acid contents. The specificity of molecular interactions together with the cooperativity of multiple proteins induce and stabilize complex membrane shapes at the mesoscale. These shapes span a wide spectrum ranging from the spherical plasma membrane to the complex cisternae of the Golgi apparatus. Mapping the relation between the protein-induced deformations at the molecular scale and the resulting mesoscale morphologies is key to bridging cellular experiments across various length scales. In this review, we focus on the theoretical and computational methods used to understand the phenomenology underlying protein-driven membrane remodeling. Interactions at the molecular scale can be computationally probed by all atom and coarse grained molecular dynamics (MD, CGMD), as well as dissipative particle dynamics (DPD) simulations, which we only describe in passing. We choose to focus on several continuum approaches extending the Canham-Helfrich elastic energy model for membranes to include the effect of curvature-inducing proteins and explore the conformational phase space of such systems. In this description, the protein is expressed in the form of a spontaneous curvature field. The approaches include field theoretical methods limited to the small deformation regime, triangulated surfaces and particle-based computational models to investigate the large-deformation regimes observed in the natural state of many biological membranes. Applications of these methods to understand the properties of biological membranes in homogeneous and inhomogeneous environments of proteins, whose underlying curvature fields are either isotropic or anisotropic, are discussed. The diversity in the curvature fields elicits a rich variety of morphological states, including tubes, discs, branched tubes, and caveola. Mapping the thermodynamic stability of these states as a function of tuning parameters such as concentration and strength of curvature induction of the proteins is discussed. The relative stabilities of these self-organized shapes are examined through free-energy calculations. The suite of methods discussed here can be tailored to applications in specific cellular settings such as endocytosis during cargo trafficking and tubulation of filopodial structures in migrating cells, which makes these methods a powerful complement to experimental studies.

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age

PubMed Central

Oiki, Shigetoshi

2015-01-01

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the ‘frozen’ crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. PMID:25833254

Channel function reconstitution and re-animation: a single-channel strategy in the postcrystal age.

PubMed

Oiki, Shigetoshi

2015-06-15

The most essential properties of ion channels for their physiologically relevant functions are ion-selective permeation and gating. Among the channel species, the potassium channel is primordial and the most ubiquitous in the biological world, and knowledge of this channel underlies the understanding of features of other ion channels. The strategy applied to studying channels changed dramatically after the crystal structure of the potassium channel was resolved. Given the abundant structural information available, we exploited the bacterial KcsA potassium channel as a simple model channel. In the postcrystal age, there are two effective frameworks with which to decipher the functional codes present in the channel structure, namely reconstitution and re-animation. Complex channel proteins are decomposed into essential functional components, and well-examined parts are rebuilt for integrating channel function in the membrane (reconstitution). Permeation and gating are dynamic operations, and one imagines the active channel by breathing life into the 'frozen' crystal (re-animation). Capturing the motion of channels at the single-molecule level is necessary to characterize the behaviour of functioning channels. Advanced techniques, including diffracted X-ray tracking, lipid bilayer methods and high-speed atomic force microscopy, have been used. Here, I present dynamic pictures of the KcsA potassium channel from the submolecular conformational changes to the supramolecular collective behaviour of channels in the membrane. These results form an integrated picture of the active channel and offer insights into the processes underlying the physiological function of the channel in the cell membrane. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

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.

Electrokinetic Response of Charge-Selective Nanostructured Polymeric Membranes

NASA Astrophysics Data System (ADS)

Schiffbauer, Jarrod; Li, Diya; Gao, Feng; Phillip, William; Chang, Hsueh-Chia

2017-11-01

Nanostructured polymeric membranes, with a tunable pore size and ease of surface molecular functionalization, are a promising material for separations, filtration, and sensing applications. Recently, such membranes have been fabricated wherein the ion selectivity is imparted by self-assembled functional groups through a two-step process. Amine groups are used to provide a positive surface charge and acid groups are used to yield a negative charge. The membranes can be fabricated as either singly-charged or patterned/mosaic membranes, where there are alternating regions of amine- lined or acid-lined pores. We demonstrate that such membranes, in addition to having many features in common with other charge selective membranes (i.e. AMX or Nafion), display a unique single-membrane rectification behavior. This is due to the asymmetric distribution of charged functional groups during the fabrication process. We demonstrate this rectification effect using both dc current-voltage characteristics as well as dc-biased electrical impedance spectroscopy. Furthermore, surface charge changes due to dc concentration polarization and generation of localized pH shifts are monitored using electrical impedance spectroscopy. (formerly at University of Notre Dame).

Structural Elucidation of the Cell-Penetrating Penetratin Peptide in Model Membranes at the Atomic Level: Probing Hydrophobic Interactions in the Blood-Brain Barrier.

PubMed

Bera, Swapna; Kar, Rajiv K; Mondal, Susanta; Pahan, Kalipada; Bhunia, Anirban

2016-09-06

Cell-penetrating peptides (CPPs) have shown promise in nonpermeable therapeutic drug delivery, because of their ability to transport a variety of cargo molecules across the cell membranes and their noncytotoxicity. Drosophila antennapedia homeodomain-derived CPP penetratin (RQIKIWFQNRRMKWKK), being rich in positively charged residues, has been increasingly used as a potential drug carrier for various purposes. Penetratin can breach the tight endothelial network known as the blood-brain barrier (BBB), permitting treatment of several neurodegenerative maladies, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. However, a detailed structural understanding of penetratin and its mechanism of action is lacking. This study defines structural features of the penetratin-derived peptide, DK17 (DRQIKIWFQNRRMKWKK), in several model membranes and describes a membrane-induced conformational transition of the DK17 peptide in these environments. A series of biophysical experiments, including high-resolution nuclear magnetic resonance spectroscopy, provides the three-dimensional structure of DK17 in different membranes mimicking the BBB or total brain lipid extract. Molecular dynamics simulations support the experimental results showing preferential binding of DK17 to particular lipids at atomic resolution. The peptide conserves the structure of the subdomain spanning residues Ile6-Arg11, despite considerable conformational variation in different membrane models. In vivo data suggest that the wild type, not a mutated sequence, enters the central nervous system. Together, these data highlight important structural and functional attributes of DK17 that could be utilized in drug delivery for neurodegenerative disorders.

Structure refinement of membrane proteins via molecular dynamics simulations.

PubMed

Dutagaci, Bercem; Heo, Lim; Feig, Michael

2018-07-01

A refinement protocol based on physics-based techniques established for water soluble proteins is tested for membrane protein structures. Initial structures were generated by homology modeling and sampled via molecular dynamics simulations in explicit lipid bilayer and aqueous solvent systems. Snapshots from the simulations were selected based on scoring with either knowledge-based or implicit membrane-based scoring functions and averaged to obtain refined models. The protocol resulted in consistent and significant refinement of the membrane protein structures similar to the performance of refinement methods for soluble proteins. Refinement success was similar between sampling in the presence of lipid bilayers and aqueous solvent but the presence of lipid bilayers may benefit the improvement of lipid-facing residues. Scoring with knowledge-based functions (DFIRE and RWplus) was found to be as good as scoring using implicit membrane-based scoring functions suggesting that differences in internal packing is more important than orientations relative to the membrane during the refinement of membrane protein homology models. © 2018 Wiley Periodicals, Inc.

[Isoflavone genistein-8-c-glycoside prevents the oxidative damages in structure and function of rat liver microsomal membranes].

PubMed

Zavodnik, L B

2003-01-01

Bioflavonoids (polyhydroxyphenols) are ubiquitous components of plants, fruits and vegetables; these compounds are efficient scavengers of free oxygen radicals and peroxides. The aim of this study was to investigate the antioxidant and radioprotective effects of genistein-8-C-glicoside (G8CG), an isoflavone, isolated from the flowers of Lipinus luteusl L. G8CG prevents dose-dependently the destruction of the cytochrome P-450 and its conversion to an inactive form cytochrome P-420, inhibits membrane lipid peroxidation and membrane SH-group oxidation in isolated rat liver microsomal membranes under tert-butylhydroperoxide-induced oxidative stress. Single whole-body gamma-irradiation (1 Gy) of rats results in blood plasma and liver microsomal membrane lipid peroxidation, impairments of microsomal membrane structure and function. Rat treatment with G8CG (75 mg/kg) developed the clear protective effect, stabilized membrane structure and improved the parameters of the monooxygenase function. We can conclude that G8CG can be used as antioxidant and radioprotective agent.

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.

Plasma membrane lipid–protein interactions affect signaling processes in sterol-biosynthesis mutants in Arabidopsis thaliana

PubMed Central

Zauber, Henrik; Burgos, Asdrubal; Garapati, Prashanth; Schulze, Waltraud X.

2014-01-01

The plasma membrane is an important organelle providing structure, signaling and transport as major biological functions. Being composed of lipids and proteins with different physicochemical properties, the biological functions of membranes depend on specific protein–protein and protein–lipid interactions. Interactions of proteins with their specific sterol and lipid environment were shown to be important factors for protein recruitment into sub-compartmental structures of the plasma membrane. System-wide implications of altered endogenous sterol levels for membrane functions in living cells were not studied in higher plant cells. In particular, little is known how alterations in membrane sterol composition affect protein and lipid organization and interaction within membranes. Here, we conducted a comparative analysis of the plasma membrane protein and lipid composition in Arabidopsis sterol-biosynthesis mutants smt1 and ugt80A2;B1. smt1 shows general alterations in sterol composition while ugt80A2;B1 is significantly impaired in sterol glycosylation. By systematically analyzing different cellular fractions and combining proteomic with lipidomic data we were able to reveal contrasting alterations in lipid–protein interactions in both mutants, with resulting differential changes in plasma membrane signaling status. PMID:24672530

Biomimetic interfaces based on S-layer proteins, lipid membranes and functional biomolecules

PubMed Central

Schuster, Bernhard; Sleytr, Uwe B.

2014-01-01

Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state-of-the-art survey of how S-layer proteins, lipids and polymers may be used as basic building blocks for the assembly of S-layer-supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and, thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas in the (lab-on-a-) biochip technology are combining composite S-layer membrane systems involving specific membrane functions with the silicon world. Thus, it might become possible to create artificial noses or tongues, where many receptor proteins have to be exposed and read out simultaneously. Moreover, S-layer-coated liposomes and emulsomes copying virus envelopes constitute promising nanoformulations for the production of novel targeting, delivery, encapsulation and imaging systems. PMID:24812051

IRSp53/BAIAP2 in dendritic spine development, NMDA receptor regulation, and psychiatric disorders.

PubMed

Kang, Jaeseung; Park, Haram; Kim, Eunjoon

2016-01-01

IRSp53 (also known as BAIAP2) is a multi-domain scaffolding and adaptor protein that has been implicated in the regulation of membrane and actin dynamics at subcellular structures, including filopodia and lamellipodia. Accumulating evidence indicates that IRSp53 is an abundant component of the postsynaptic density at excitatory synapses and an important regulator of actin-rich dendritic spines. In addition, IRSp53 has been implicated in diverse psychiatric disorders, including autism spectrum disorders, schizophrenia, and attention deficit/hyperactivity disorder. Mice lacking IRSp53 display enhanced NMDA (N-methyl-d-aspartate) receptor function accompanied by social and cognitive deficits, which are reversed by pharmacological suppression of NMDA receptor function. These results suggest the hypothesis that defective actin/membrane modulation in IRSp53-deficient dendritic spines may lead to social and cognitive deficits through NMDA receptor dysfunction. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Ion transport across the biological membrane by computational protein design

NASA Astrophysics Data System (ADS)

Grigoryan, Gevorg

The cellular membrane is impermeable to most of the chemicals the cell needs to take in or discard to survive. Therefore, transporters-a class of transmembrane proteins tasked with shuttling cargo chemicals in and out of the cell-are essential to all cellular life. From existing crystal structures, we know transporters to be complex machines, exquisitely tuned for specificity and controllability. But how could membrane-bound life have evolved if it needed such complex machines to exist first? To shed light onto this question, we considered the task of designing a transporter de novo. As our guiding principle, we took the ``alternating-access model''-a conceptual mechanism stating that transporters work by rocking between two conformations, each exposing the cargo-binding site to either the intra- or the extra-cellular environment. A computational design framework was developed to encode an anti-parallel four-helix bundle that rocked between two alternative states to orchestrate the movement of Zn(II) ions across the membrane. The ensemble nature of both states was accounted for using a free energy-based approach, and sequences were chosen based on predicted formation of the targeted topology in the membrane and bi-stability. A single sequence was prepared experimentally and shown to function as a Zn(II) transporter in lipid vesicles. Further, transport was specific to Zn(II) ions and several control peptides supported the underlying design principles. This included a mutant designed to retain all properties but with reduced rocking, which showed greatly depressed transport ability. These results suggest that early transporters could have evolved in the context of simple topologies, to be later tuned by evolution for improved properties and controllability. Our study also serves as an important advance in computational protein design, showing the feasibility of designing functional membrane proteins and of tuning conformational landscapes for desired function. Alfred P. Sloan Foundation Research Fellowship.

Sputter-deposited fuel cell membranes and electrodes

NASA Technical Reports Server (NTRS)

Narayanan, Sekharipuram R. (Inventor); Jeffries-Nakamura, Barbara (Inventor); Chun, William (Inventor); Ruiz, Ron P. (Inventor); Valdez, Thomas I. (Inventor)

2001-01-01

A method for preparing a membrane for use in a fuel cell membrane electrode assembly includes the steps of providing an electrolyte membrane, and sputter-depositing a catalyst onto the electrolyte membrane. The sputter-deposited catalyst may be applied to multiple sides of the electrolyte membrane. A method for forming an electrode for use in a fuel cell membrane electrode assembly includes the steps of obtaining a catalyst, obtaining a backing, and sputter-depositing the catalyst onto the backing. The membranes and electrodes are useful for assembling fuel cells that include an anode electrode, a cathode electrode, a fuel supply, and an electrolyte membrane, wherein the electrolyte membrane includes a sputter-deposited catalyst, and the sputter-deposited catalyst is effective for sustaining a voltage across a membrane electrode assembly in the fuel cell.

Surface Functionalization of Polymeric Nanoparticles with Umbilical Cord-Derived Mesenchymal Stem Cell Membrane for Tumor-Targeted Therapy.

PubMed

Yang, Na; Ding, Yanping; Zhang, Yinlong; Wang, Bin; Zhao, Xiao; Cheng, Keman; Huang, Yixin; Taleb, Mohammad; Zhao, Jing; Dong, Wen-Fei; Zhang, Lirong; Nie, Guangjun

2018-06-15

Multiple cell plasma membranes have been utilized for surface functionalization of synthetic nanomaterials and construction of biomimetic drug delivery systems for cancer treatment. The natural characters and facile isolation of original cells facilitate the biomedical applications of plasma membranes in functionalizing nanocarriers. Human umbilical cord-derived mesenchymal stem cells (MSC) have been identified to show tropism towards malignant lesions and have great advantages in ease of acquisition, low immunogenicity, and high proliferative ability. Here we developed a poly(lactic-co-glycolic acid) (PLGA) nanoparticle with a layer of plasma membrane from umbilical cord MSC coating on the surface for tumor-targeted delivery of chemotherapy. Functionalization of MSC plasma membrane significantly enhanced the cellular uptake efficiency of PLGA nanoparticles, the tumor cell killing efficacy of PLGA-encapsulated doxorubicin, and most importantly the tumor-targeting and accumulation of the nanoparticles. As a result, this MSC-mimicking nanoformulation led to remarkable tumor growth inhibition and induced obvious apoptosis within tumor lesions. This study for the first time demonstrated the great potential of umbilical cord MSC plasma membranes in functionalizing nanocarriers with inherent tumor-homing features, and the high feasibility of such biomimetic nanoformulations in cancer therapy.

Superresolution imaging of viral protein trafficking

PubMed Central

Salka, Kyle; Bhuvanendran, Shivaprasad; Yang, David

2015-01-01

The endoplasmic reticulum (ER) membrane is closely apposed to the outer mitochondrial membrane (OMM), which facilitates communication between these organelles. These contacts, known as mitochondria-associated membranes (MAM), facilitate calcium signaling, lipid transfer, as well as antiviral and stress responses. How cellular proteins traffic to the MAM, are distributed therein, and interact with ER and mitochondrial proteins are subject of great interest. The human cytomegalovirus UL37 exon 1 protein or viral mitochondria-localized inhibitor of apoptosis (vMIA) is crucial for viral growth. Upon synthesis at the ER, vMIA traffics to the MAM and OMM, where it reprograms the organization and function of these compartments. vMIA significantly changes the abundance of cellular proteins at the MAM and OMM, including proteins that regulate calcium homeostasis and cell death. Through the use of superresolution imaging, we have shown that vMIA is distributed at the OMM in nanometer scale clusters. This is similar to the clusters reported for the mitochondrial calcium channel, VDAC, as well as electron transport chain, translocase of the OMM complex, and mitochondrial inner membrane organizing system components. Thus, aside from addressing how vMIA targets the MAM and regulates survival of infected cells, biochemical studies and superresolution imaging of vMIA offer insights into the formation, organization, and functioning of MAM. Here, we discuss these insights into trafficking, function, and organization of vMIA at the MAM and OMM and discuss how the use of superresolution imaging is contributing to the study of the formation and trafficking of viruses. PMID:25724304

Mesoporous fluorocarbon-modified silica aerogel membranes enabling long-term continuous CO2 capture with large absorption flux enhancements.

PubMed

Lin, Yi-Feng; Chen, Chien-Hua; Tung, Kuo-Lun; Wei, Te-Yu; Lu, Shih-Yuan; Chang, Kai-Shiun

2013-03-01

The use of a membrane contactor combined with a hydrophobic porous membrane and an amine absorbent has attracted considerable attention for the capture of CO2 because of its extensive use, low operational costs, and low energy consumption. The hydrophobic porous membrane interface prevents the passage of the amine absorbent but allows the penetration of CO2 molecules that are captured by the amine absorbent. Herein, highly porous SiO2 aerogels modified with hydrophobic fluorocarbon functional groups (CF3 ) were successfully coated onto a macroporous Al2 O3 membrane; their performance in a membrane contactor for CO2 absorption is discussed. The SiO2 aerogel membrane modified with CF3 functional groups exhibits the highest CO2 absorption flux and can be continuously operated for CO2 absorption for extended periods of time. This study suggests that a SiO2 aerogel membrane modified with CF3 functional groups could potentially be used in a membrane contactor for CO2 absorption. Also, the resulting hydrophobic SiO2 aerogel membrane contactor is a promising technology for large-scale CO2 absorption during the post-combustion process in power plants. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effects of pressure and electrical charge on macromolecular transport across bovine lens basement membrane.

PubMed

Ferrell, Nicholas; Cameron, Kathleen O; Groszek, Joseph J; Hofmann, Christina L; Li, Lingyan; Smith, Ross A; Bian, Aihua; Shintani, Ayumi; Zydney, Andrew L; Fissell, William H

2013-04-02

Molecular transport through the basement membrane is important for a number of physiological functions, and dysregulation of basement membrane architecture can have serious pathological consequences. The structure-function relationships that govern molecular transport in basement membranes are not fully understood. The basement membrane from the lens capsule of the eye is a collagen IV-rich matrix that can easily be extracted and manipulated in vitro. As such, it provides a convenient model for studying the functional relationships that govern molecular transport in basement membranes. Here we investigate the effects of increased transmembrane pressure and solute electrical charge on the transport properties of the lens basement membrane (LBM) from the bovine eye. Pressure-permeability relationships in LBM transport were governed primarily by changes in diffusive and convective contributions to solute flux and not by pressure-dependent changes in intrinsic membrane properties. The solute electrical charge had a minimal but statistically significant effect on solute transport through the LBM that was opposite of the expected electrokinetic behavior. The observed transport characteristics of the LBM are discussed in the context of established membrane transport modeling and previous work on the effects of pressure and electrical charge in other basement membrane systems. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

An Integrated Framework Advancing Membrane Protein Modeling and Design

PubMed Central

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

2015-01-01

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

Plant glycosylphosphatidylinositol (GPI) anchored proteins at the plasma membrane-cell wall nexus.

PubMed

Yeats, Trevor H; Bacic, Antony; Johnson, Kim L

2018-04-18

Approximately 1% of plant proteins are predicted to be post-translationally modified with a glycosylphosphatidylinositol (GPI) anchor that tethers the polypeptide to the outer leaflet of the plasma membrane. While the synthesis and structure of GPI anchors is largely conserved across eukaryotes, the repertoire of functional domains present in the GPI-anchored proteome has diverged substantially. In plants, this includes a large fraction of the GPI-anchored proteome being further modified with plant-specific arabinogalactan (AG) O-glycans. The importance of the GPI-anchored proteome to plant development is underscored by the fact that GPI biosynthetic null mutants exhibit embryo lethality. Mutations in genes encoding specific GPI-anchored proteins (GAPs) further supports their contribution to diverse biological processes occurring at the interface of the plasma membrane and cell wall, including signaling, cell wall metabolism, cell wall polymer cross-linking, and plasmodesmatal transport. Here, we review the literature concerning plant GPI-anchored proteins in the context of their potential to act as molecular hubs that mediate interactions between the plasma membrane and the cell wall and their potential to transduce the signal into the protoplast and thereby activate signal transduction pathways. This article is protected by copyright. All rights reserved.

Nanomaterials for Space Exploration Applications

NASA Technical Reports Server (NTRS)

Moloney, Padraig G.

2006-01-01

Nano-engineered materials are multi-functional materials with superior mechanical, thermal and electrical properties. Nanomaterials may be used for a variety of space exploration applications, including ultracapacitors, active/passive thermal management materials, and nanofiltration for water recovery. Additional applications include electrical power/energy storage systems, hybrid systems power generation, advanced proton exchange membrane fuel cells, and air revitalization. The need for nanomaterials and their growth, characterization, processing and space exploration applications is discussed. Data is presented for developing solid-supported amine adsorbents based on carbon nanotube materials and functionalization of nanomaterials is examined.

Spontaneous formation of structurally diverse membrane channel architectures from a single antimicrobial peptide

NASA Astrophysics Data System (ADS)

Wang, Yukun; Chen, Charles H.; Hu, Dan; Ulmschneider, Martin B.; Ulmschneider, Jakob P.

2016-11-01

Many antimicrobial peptides (AMPs) selectively target and form pores in microbial membranes. However, the mechanisms of membrane targeting, pore formation and function remain elusive. Here we report an experimentally guided unbiased simulation methodology that yields the mechanism of spontaneous pore assembly for the AMP maculatin at atomic resolution. Rather than a single pore, maculatin forms an ensemble of structurally diverse temporarily functional low-oligomeric pores, which mimic integral membrane protein channels in structure. These pores continuously form and dissociate in the membrane. Membrane permeabilization is dominated by hexa-, hepta- and octamers, which conduct water, ions and small dyes. Pores form by consecutive addition of individual helices to a transmembrane helix or helix bundle, in contrast to current poration models. The diversity of the pore architectures--formed by a single sequence--may be a key feature in preventing bacterial resistance and could explain why sequence-function relationships in AMPs remain elusive.

Functional dynamics of cell surface membrane proteins

NASA Astrophysics Data System (ADS)

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

Ion transport controlled by nanoparticle-functionalized membranes

NASA Astrophysics Data System (ADS)

Barry, Edward; McBride, Sean P.; Jaeger, Heinrich M.; Lin, Xiao-Min

2014-12-01

From proton exchange membranes in fuel cells to ion channels in biological membranes, the well-specified control of ionic interactions in confined geometries profoundly influences the transport and selectivity of porous materials. Here we outline a versatile new approach to control a membrane’s electrostatic interactions with ions by depositing ligand-coated nanoparticles around the pore entrances. Leveraging the flexibility and control by which ligated nanoparticles can be synthesized, we demonstrate how ligand terminal groups such as methyl, carboxyl and amine can be used to tune the membrane charge density and control ion transport. Further functionality, exploiting the ligands as binding sites, is demonstrated for sulfonate groups resulting in an enhancement of the membrane charge density. We then extend these results to smaller dimensions by systematically varying the underlying pore diameter. As a whole, these results outline a previously unexplored method for the nanoparticle functionalization of membranes using ligated nanoparticles to control ion transport.

Bacterial Reaction Centers Purified with Styrene Maleic Acid Copolymer Retain Native Membrane Functional Properties and Display Enhanced Stability**

PubMed Central

Swainsbury, David J K; Scheidelaar, Stefan; van Grondelle, Rienk; Killian, J Antoinette; Jones, Michael R

2014-01-01

Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opportunity to compare the properties of an integral membrane protein in different artificial lipid/surfactant environments with those in the native bilayer. Here, we demonstrate that reaction centers purified using a styrene maleic acid copolymer remain associated with a complement of native lipids and do not display the modified functional properties that typically result from detergent solubilization. Direct comparisons show that reaction centers are more stable in this copolymer/lipid environment than in a detergent micelle or even in the native membrane, suggesting a promising new route to exploitation of such photovoltaic integral membrane proteins in device applications. PMID:25212490

Functional dynamics of cell surface membrane proteins.

PubMed

Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

2014-04-01

Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

Functionality in Electrospun Nanofibrous Membranes Based on Fiber's Size, Surface Area, and Molecular Orientation

PubMed Central

Matsumoto, Hidetoshi; Tanioka, Akihiko

2011-01-01

Electrospinning is a versatile method for forming continuous thin fibers based on an electrohydrodynamic process. This method has the following advantages: (i) the ability to produce thin fibers with diameters in the micrometer and nanometer ranges; (ii) one-step forming of the two- or three-dimensional nanofiber network assemblies (nanofibrous membranes); and (iii) applicability for a broad spectrum of molecules, such as synthetic and biological polymers and polymerless sol-gel systems. Electrospun nanofibrous membranes have received significant attention in terms of their practical applications. The major advantages of nanofibers or nanofibrous membranes are the functionalities based on their nanoscaled-size, highly specific surface area, and highly molecular orientation. These functionalities of the nanofibrous membranes can be controlled by their fiber diameter, surface chemistry and topology, and internal structure of the nanofibers. This report focuses on our studies and describes fundamental aspects and applications of electrospun nanofibrous membranes. PMID:24957735

Measuring and Modeling Sonoporation Dynamics in Mammalian Cells via Calcium Imaging

NASA Astrophysics Data System (ADS)

Kumon, R. E.; Parikh, P.; Sabens, D.; Aehle, M.; Kourennyi, D.; Deng, C. X.

2007-05-01

In this study, calcium imaging via the fluorescent indicator Fura-2 is used to characterize the sonoporation of Chinese Hamster Ovarian (CHO) cells in the presence of Optison™ microbubbles. Evolution of the calcium concentration within cells is determined from real-time fluorescence intensity measurements before, during, and after exposure to a 1 MHz ultrasound tone burst (0.2 s, 0.45 MPa). To relate microscopic sonoporation parameters to the measurements, an analytical model that includes sonoporation and plasma membrane transport is developed, assuming rapid mixing (uniform spatial distribution) in the cell. Fitting the measured data to the model provides estimated values for the poration area as a function of poration relaxation rate as well as plasma membrane pump and leakage rates. A modified compartment model that includes the effects of sonoporation, buffering proteins, and transport across the plasma membrane, endoplasmic reticulum, and mitochondria is also investigated. Numerical 3solutions of this model show a variety of behaviors for the calcium dynamics of the cell.

Membrane-Based Functions in the Origin of Cellular Life

NASA Technical Reports Server (NTRS)

Chipot, Christophe; New, Michael H.; Schweighofer, Karl; Pohorille, Andrew; Wilson, Michael A.

1999-01-01

Our objective is to help explain how the earliest ancestors of contemporary cells (protocells) performed their essential functions employing only the molecules available in the protobiological milieu. Our hypothesis is that vesicles, built of amphiphilic, membrane-forming materials, emerged early in protobiological evolution and served as precursors to protocells. We further assume that the cellular functions associated with contemporary membranes, such as capturing and, transducing of energy, signaling, or sequestering organic molecules and ions, evolved in these membrane environments. An alternative hypothesis is that these functions evolved in different environments and were incorporated into membrane-bound structures at some later stage of evolution. We focus on the application of the fundamental principles of physics and chemistry to determine how they apply to the formation of a primitive, functional cell. Rather than attempting to develop specific models for cellular functions and to identify the origin of the molecules which perform these functions, our goal is to define the structural and energetic conditions that any successful model must fulfill, therefore providing physico-chemical boundaries for these models. We do this by carrying out large-scale, molecular level computer simulations on systems of interest.

Putative terminator and/or effector functions of Arf GAPs in the trafficking of clathrin-coated vesicles.

PubMed

Kon, Shunsuke; Funaki, Tomo; Satake, Masanobu

2011-05-01

The role of ArfGAP1 as a terminator or effector in COPi-vesicle formation has been the subject of ongoing discussions. Here, the discussion on the putative terminator/effector functions has been enlarged to include Arf GAP members involved in the formation of clathrin-coated vesicles. ACAP1, whose role has been studied extensively, enhances the recycling of endocytosed proteins to the plasma membrane. Importantly, this positive role appears to be an overall reflection of both the terminator and effector activities attributed to ACAP1. Other Arf GAP subtypes have also been suggested to possess both terminator and effector activities. Interestingly, while most Arf GAP proteins regulate membrane trafficking by acting as facilitators, a few Arf GAP subtypes act as inhibitors.

KCNE Regulation of K+ Channel Trafficking – a Sisyphean Task?

PubMed Central

Kanda, Vikram A.; Abbott, Geoffrey W.

2012-01-01

Voltage-gated potassium (Kv) channels shape the action potentials of excitable cells and regulate membrane potential and ion homeostasis in excitable and non-excitable cells. With 40 known members in the human genome and a variety of homomeric and heteromeric pore-forming α subunit interactions, post-translational modifications, cellular locations, and expression patterns, the functional repertoire of the Kv α subunit family is monumental. This versatility is amplified by a host of interacting proteins, including the single membrane-spanning KCNE ancillary subunits. Here, examining both the secretory and the endocytic pathways, we review recent findings illustrating the surprising virtuosity of the KCNE proteins in orchestrating not just the function, but also the composition, diaspora and retrieval of channels formed by their Kv α subunit partners. PMID:22754540