Localized ridge defect augmentation using human pericardium membrane and demineralized bone matrix.

PubMed

Vidyadharan, Arun Kumar; Ravindran, Anjana

2014-01-01

Patient wanted to restore her lost teeth with implants in the lower left first molar and second premolar region. Cone beam computerized tomography (CBCT) revealed inadequate bone width and height around future implant sites. The extraction socket of second premolar area revealed inadequate socket healing with sparse bone fill after 4 months of extraction. To evaluate the clinical feasibility of using a collagen physical resorbable barrier made of human pericardium (HP) to augment localized alveolar ridge defects for the subsequent placement of dental implants. Ridge augmentation was done in the compromised area using Puros® demineralized bone matrix (DBM) Putty with chips and an HP allograft membrane. Horizontal (width) and vertical hard tissue measurements with CBCT were recorded on the day of ridge augmentation surgery, 4 month and 7 months follow-up. Intra oral periapical taken 1 year after implant installation showed minimal crestal bone loss. Bone volume achieved through guided bone regeneration was a gain of 4.8 mm horizontally (width) and 6.8 mm vertically in the deficient ridge within a period of 7 months following the procedure. The results suggested that HP Allograft membrane may be a suitable component for augmentation of localized alveolar ridge defects in conjunction with DBM with bone chips.

Membrane raft association is a determinant of plasma membrane localization

PubMed Central

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

2014-01-01

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting. PMID:24912166

Wood cell-wall structure requires local 2D-microtubule disassembly by a novel plasma membrane-anchored protein.

PubMed

Oda, Yoshihisa; Iida, Yuki; Kondo, Yuki; Fukuda, Hiroo

2010-07-13

Plant cells have evolved cortical microtubules, in a two-dimensional space beneath the plasma membrane, that regulate patterning of cellulose deposition. Although recent studies have revealed that several microtubule-associated proteins facilitate self-organization of transverse cortical microtubules, it is still unknown how diverse patterns of cortical microtubules are organized in different xylem cells, which are the major components of wood. Using our newly established in vitro xylem cell differentiation system, we found that a novel microtubule end-tracking protein, microtubule depletion domain 1 (MIDD1), was anchored to distinct plasma membrane domains and promoted local microtubule disassembly, resulting in pits on xylem cell walls. The introduction of RNA interference for MIDD1 resulted in the failure of local microtubule depletion and the formation of secondary walls without pits. Conversely, the overexpression of MIDD1 reduced microtubule density. MIDD1 has two coiled-coil domains for the binding to microtubules and for the anchorage to plasma membrane domains, respectively. Combination of the two coils caused end tracking of microtubules during shrinkage and suppressed their rescue events. Our results indicate that MIDD1 integrates spatial information in the plasma membrane with cortical microtubule dynamics for determining xylem cell wall pattern. Copyright 2010 Elsevier Ltd. All rights reserved.

Architecture, component, and microbiome of biofilm involved in the fouling of membrane bioreactors.

PubMed

Inaba, Tomohiro; Hori, Tomoyuki; Aizawa, Hidenobu; Ogata, Atsushi; Habe, Hiroshi

2017-01-01

Biofilm formation on the filtration membrane and the subsequent clogging of membrane pores (called biofouling) is one of the most persistent problems in membrane bioreactors for wastewater treatment and reclamation. Here, we investigated the structure and microbiome of fouling-related biofilms in the membrane bioreactor using non-destructive confocal reflection microscopy and high-throughput Illumina sequencing of 16S rRNA genes. Direct confocal reflection microscopy indicated that the thin biofilms were formed and maintained regardless of the increasing transmembrane pressure, which is a common indicator of membrane fouling, at low organic-loading rates. Their solid components were primarily extracellular polysaccharides and microbial cells. In contrast, high organic-loading rates resulted in a rapid increase in the transmembrane pressure and the development of the thick biofilms mainly composed of extracellular lipids. High-throughput sequencing revealed that the biofilm microbiomes, including major and minor microorganisms, substantially changed in response to the organic-loading rates and biofilm development. These results demonstrated for the first time that the architectures, chemical components, and microbiomes of the biofilms on fouled membranes were tightly associated with one another and differed considerably depending on the organic-loading conditions in the membrane bioreactor, emphasizing the significance of alternative indicators other than the transmembrane pressure for membrane biofouling.

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

PubMed

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

2008-02-01

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

Membrane permeable local anesthetics modulate NaV1.5 mechanosensitivity

PubMed Central

Beyder, Arthur; Strege, Peter R.; Bernard, Cheryl; Farrugia, Gianrico

2012-01-01

Voltage-gated sodium selective ion channel NaV1.5 is expressed in the heart and the gastrointestinal tract, which are mechanically active organs. NaV1.5 is mechanosensitive at stimuli that gate other mechanosensitive ion channels. Local anesthetic and antiarrhythmic drugs act upon NaV1.5 to modulate activity by multiple mechanisms. This study examined whether NaV1.5 mechanosensitivity is modulated by local anesthetics. NaV1.5 channels wereexpressed in HEK-293 cells, and mechanosensitivity was tested in cell-attached and excised inside-out configurations. Using a novel protocol with paired voltage ladders and short pressure pulses, negative patch pressure (-30 mmHg) in both configurations produced a hyperpolarizing shift in the half-point of the voltage-dependence of activation (V1/2a) and inactivation (V1/2i) by about -10 mV. Lidocaine (50 µM) inhibited the pressure-induced shift of V1/2a but not V1/2i. Lidocaine inhibited the tonic increase in pressure-induced peak current in a use-dependence protocol, but it did not otherwise affect use-dependent block. The local anesthetic benzocaine, which does not show use-dependent block, also effectively blocked a pressure-induced shift in V1/2a. Lidocaine inhibited mechanosensitivity in NaV1.5 at the local anesthetic binding site mutated (F1760A). However, a membrane impermeable lidocaine analog QX-314 did not affect mechanosensitivity of F1760A NaV1.5 when applied from either side of the membrane. These data suggest that the mechanism of lidocaine inhibition of the pressure-induced shift in the half-point of voltage-dependence of activation is separate from the mechanisms of use-dependent block. Modulation of NaV1.5 mechanosensitivity by the membrane permeable local anesthetics may require hydrophobic access and may involve membrane-protein interactions. PMID:22874086

Electric field-induced reorganization of two-component supported bilayer membranes

PubMed Central

Groves, Jay T.; Boxer, Steven G.; McConnell, Harden M.

1997-01-01

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory’s approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi–Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes. PMID:9391034

Electric field-induced reorganization of two-component supported bilayer membranes.

PubMed

Groves, J T; Boxer, S G; McConnell, H M

1997-12-09

Application of electric fields tangent to the plane of a confined patch of fluid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory's approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi-Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine- and cardiolipin-containing membranes.

p27Kip1 localizes to detergent-insoluble microdomains within lymphocyte membranes.

PubMed Central

Yaroslavskiy, B. B.; Stolz, D. B.; Watkins, S. C.; Alber, S. M.; Bradbury, N. A.; Steinman, R. A.

2001-01-01

BACKGROUND: Low levels of the cyclin-dependent kinase inhibitor p27Kip1 are associated with poor prognosis in cancer. It is unclear whether this is related strictly to p27Kip1-mediated cell cycle inhibition or to other, possibly extranuclear, roles of this protein. In this study, we examined p27Kip1 expression in quiescent and activated lymphocytes. T-cell membranes have been shown to possess sphingolipid and cholesterol-rich microdomains that are insoluble in non-ionic detergents. These "rafts" provide a scaffold for signaling proteins. Signal transduction coincides with coalescence of these microdomains into larger complexes. METHODS: Localization of p27Kip1 was studied by electron and confocal microscopy. Association of p27Kip1 with membrane microdomains in unstimulated and stimulated lymphocytes was determined using Western blots analysis of isolated membranes variably treated with detergents. RESULTS: We demonstrated that p27Kip1 was present in clusters associated with the plasma membrane in normal lymphocytes. The solubility profile of p27Kip1 in isolated membranes indicated that it was localized to raft structures. When lymphocytes were stimulated, however, p27Kip1 was excluded from aggregated raft complexes. CONCLUSIONS: This study identifies, for the first time, the localization of p27 within a membrane microdomain associated with signaling. Because some cell surface signaling complexes lose p27Kip1 upon cellular activation, p27Kip1 may play a functional role in modulating membrane signaling. PMID:11474127

Vitreoscilla hemoglobin. Intracellular localization and binding to membranes.

PubMed

Ramandeep; Hwang, K W; Raje, M; Kim, K J; Stark, B C; Dikshit, K L; Webster, D A

2001-07-06

The obligate aerobic bacterium, Vitreoscilla, synthesizes elevated quantities of a homodimeric hemoglobin (VHb) under hypoxic growth conditions. Expression of VHb in heterologous hosts often enhances growth and product formation. A role in facilitating oxygen transfer to the respiratory membranes is one explanation of its cellular function. Immunogold labeling of VHb in both Vitreoscilla and recombinant Escherichia coli bearing the VHb gene clearly indicated that VHb has a cytoplasmic (not periplasmic) localization and is concentrated near the periphery of the cytosolic face of the cell membrane. OmpA signal-peptide VHb fusions were transported into the periplasm in E. coli, but this did not confer any additional growth advantage. The interaction of VHb with respiratory membranes was also studied. The K(d) values for the binding of VHb to Vitreoscilla and E. coli cell membranes were approximately 5-6 microm, a 4-8-fold higher affinity than those of horse myoglobin and hemoglobin for these same membranes. VHb stimulated the ubiquinol-1 oxidase activity of inverted Vitreoscilla membranes by 68%. The inclusion of Vitreoscilla cytochrome bo in proteoliposomes led to 2.4- and 6-fold increases in VHb binding affinity and binding site number, respectively, relative to control liposomes, suggesting a direct interaction between VHb and cytochrome bo.

Partitioning behavior of aromatic components in jet fuel into diverse membrane-coated fibers.

PubMed

Baynes, Ronald E; Xia, Xin-Rui; Barlow, Beth M; Riviere, Jim E

2007-11-01

Jet fuel components are known to partition into skin and produce occupational irritant contact dermatitis (OICD) and potentially adverse systemic effects. The purpose of this study was to determine how jet fuel components partition (1) from solvent mixtures into diverse membrane-coated fibers (MCFs) and (2) from biological media into MCFs to predict tissue distribution. Three diverse MCFs, polydimethylsiloxane (PDMS, lipophilic), polyacrylate (PA, polarizable), and carbowax (CAR, polar), were selected to simulate the physicochemical properties of skin in vivo. Following an appropriate equilibrium time between the MCF and dosing solutions, the MCF was injected directly into a gas chromatograph/mass spectrometer (GC-MS) to quantify the amount that partitioned into the membrane. Three vehicles (water, 50% ethanol-water, and albumin-containing media solution) were studied for selected jet fuel components. The more hydrophobic the component, the greater was the partitioning into the membranes across all MCF types, especially from water. The presence of ethanol as a surrogate solvent resulted in significantly reduced partitioning into the MCFs with discernible differences across the three fibers based on their chemistries. The presence of a plasma substitute (media) also reduced partitioning into the MCF, with the CAR MCF system being better correlated to the predicted partitioning of aromatic components into skin. This study demonstrated that a single or multiple set of MCF fibers may be used as a surrogate for octanol/water systems and skin to assess partitioning behavior of nine aromatic components frequently formulated with jet fuels. These diverse inert fibers were able to assess solute partitioning from a blood substitute such as media into a membrane possessing physicochemical properties similar to human skin. This information may be incorporated into physiologically based pharmacokinetic (PBPK) models to provide a more accurate assessment of tissue dosimetry of

Plasma membrane localization of multidrug resistance-associated protein homologs in brain capillary endothelial cells.

PubMed

Zhang, Yan; Schuetz, John D; Elmquist, William F; Miller, Donald W

2004-11-01

Several multidrug resistance-associated protein (MRP) homologs are expressed in brain microvessel endothelial cells forming the blood-brain barrier (BBB). The influence of these MRP transporters on BBB permeability will be dependent on their localization within the brain microvessel endothelial cells. Using two different and complementary approaches, the localization of various MPR homologs (MRP1, MRP4, and MRP5) was examined in primary cultured bovine brain microvessel endothelial cells (BBMECs). The first approach involved centrifugal separation of apical and basolateral plasma membranes of cultured BBMECs. The membrane fractions were then subjected to Western blot analysis for MRPs. The second approach used confocal laser scanning microscopy to determine membrane localization of MRPs in BBMECs. Results show a predominantly apical plasma membrane distribution for MRP1 and MRP5, and an almost equal distribution of MRP4 on the apical and basolateral plasma membrane of BBMECs. These studies provide the first demonstration of the localization of MRP1, MRP4, and MRP5 homologs in brain microvessel endothelial cells. The present studies also indicate that the localization of MRPs in the endothelial cells forming the BBB is different from that observed in polarized epithelial cells and thus may contribute to the reduced entry and enhanced elimination of organic anions and nucleotides in the brain.

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

Spitzenkorper, exocyst, and polarisome components in Candida albicans hyphae show different patterns of localization and have distinct dynamic properties.

PubMed

Jones, Laura A; Sudbery, Peter E

2010-10-01

During the extreme polarized growth of fungal hyphae, secretory vesicles are thought to accumulate in a subapical region called the Spitzenkörper. The human fungal pathogen Candida albicans can grow in a budding yeast or hyphal form. When it grows as hyphae, Mlc1 accumulates in a subapical spot suggestive of a Spitzenkörper-like structure, while the polarisome components Spa2 and Bud6 localize to a surface crescent. Here we show that the vesicle-associated protein Sec4 also localizes to a spot, confirming that secretory vesicles accumulate in the putative C. albicans Spitzenkörper. In contrast, exocyst components localize to a surface crescent. Using a combination of fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) experiments and cytochalasin A to disrupt actin cables, we showed that Spitzenkörper-located proteins are highly dynamic. In contrast, exocyst and polarisome components are stably located at the cell surface. It is thought that in Saccharomyces cerevisiae exocyst components are transported to the cell surface on secretory vesicles along actin cables. If each vesicle carried its own complement of exocyst components, then it would be expected that exocyst components would be as dynamic as Sec4 and would have the same pattern of localization. This is not what we observe in C. albicans. We propose a model in which a stream of vesicles arrives at the tip and accumulates in the Spitzenkörper before onward delivery to the plasma membrane mediated by exocyst and polarisome components that are more stable residents of the cell surface.

Cardiolipin Synthesis and Outer Membrane Localization Are Required for Shigella flexneri Virulence.

PubMed

Rossi, Rachael M; Yum, Lauren; Agaisse, Hervé; Payne, Shelley M

2017-08-29

Cardiolipin, an anionic phospholipid that resides at the poles of the inner and outer membranes, is synthesized primarily by the putative cardiolipin synthase ClsA in Shigella flexneri An S. flexneri clsA mutant had no cardiolipin detected within its membrane, grew normally in vitro , and invaded cultured epithelial cells, but it failed to form plaques in epithelial cell monolayers, indicating that cardiolipin is required for virulence. The clsA mutant was initially motile within the host cell cytoplasm but formed filaments and lost motility during replication and failed to spread efficiently to neighboring cells. Mutation of pbgA , which encodes the transporter for cardiolipin from the inner membrane to the outer membrane, also resulted in loss of plaque formation. The S. flexneri pbgA mutant had normal levels of cardiolipin in the inner membrane, but no cardiolipin was detected in the outer membrane. The pbgA mutant invaded and replicated normally within cultured epithelial cells but failed to localize the actin polymerization protein IcsA properly on the bacterial surface and was unable to spread to neighboring cells. The clsA mutant, but not the pbgA mutant, had increased phosphatidylglycerol in the outer membrane. This appeared to compensate partially for the loss of cardiolipin in the outer membrane, allowing some IcsA localization in the outer membrane of the clsA mutant. We propose a dual function for cardiolipin in S. flexneri pathogenesis. In the inner membrane, cardiolipin is essential for proper cell division during intracellular growth. In the outer membrane, cardiolipin facilitates proper presentation of IcsA on the bacterial surface. IMPORTANCE The human pathogen Shigella flexneri causes bacterial dysentery by invading colonic epithelial cells, rapidly multiplying within their cytoplasm, and then spreading intercellularly to neighboring cells. Worldwide, Shigella spp. infect hundreds of millions of people annually, with fatality rates up to 15

Dishevelled-induced phosphorylation regulates membrane localization of Par1b

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

Terabayashi, Takeshi; Funato, Yosuke; Miki, Hiroaki, E-mail: hmiki@protein.osaka-u.ac.jp

2008-10-31

Par1b is an evolutionarily conserved kinase that plays crucial roles in cell polarity. Controlling intracellular localization of Par1b is important for its biological activity. We previously reported that Wnt stimulation or expression of Dvl promotes accumulation of Par1b in the membrane (T. Terabayashi, T.J. Itoh, H. Yamaguchi, Y. Yoshimura, Y. Funato, S. Ohno, H. Miki, Polarity-Regulating Kinase Partitioning-Defective 1/Microtubule Affinity-Regulating Kinase 2 Negatively Regulates Development of Dendrites on Hippocampal Neurons, J. Neurosci. 27 (2007) 13098-13107). However, its molecular mechanism remains unclear. Here we show the importance of Par1b phosphorylation in the regulation of membrane localization. We find that Thr-324 ismore » phosphorylated in a Dvl-dependent manner. Interestingly, the conversion of Thr-324 to Glu results in a significant accumulation of Par1b in the membrane, without any effects on the kinase activity. Moreover, the phospho-mimicking Par1b mutant does not antagonistically function against Dvl in microtubule stabilization and neurite extension, although wildtype Par1b does. These results suggest that membrane accumulation of Par1b induced by Dvl is regulated by its phosphorylation status, which is important for Par1b to regulate the microtubule dynamics.« less

Membrane curvature and the Tol-Pal complex determine polar localization of the chemoreceptor Tar in E. coli.

PubMed

Saaki, Terrens N V; Strahl, Henrik; Hamoen, Leendert W

2018-02-20

Chemoreceptors are localized at the cell poles of Escherichia coli and other rod-shaped bacteria. Over the years different mechanisms have been put forward to explain this polar localization; from stochastic clustering, membrane curvature driven localization, interactions with the Tol-Pal complex, to nucleoid exclusion. To evaluate these mechanisms, we monitored the cellular localization of the aspartate chemoreceptor Tar in different deletion mutants. We did not find any indication for either stochastic cluster formation or nucleoid exclusion. However, the presence of a functional Tol-Pal complex appeared to be essential to retain Tar at cell poles. Interestingly, Tar still accumulated at midcell in tol and in pal deletion mutants. In these mutants, the protein appears to gather at the base of division septa, a region characterised by strong membrane curvature. Chemoreceptors, like Tar, form trimer-of-dimers that bend the cell membrane due to a rigid tripod structure. The curvature approaches the curvature of the cell membrane generated during cell division, and localization of chemoreceptor tripods at curved membrane areas is therefore energetically favourable as it lowers membrane tension. Indeed, when we introduced mutations in Tar that abolish the rigid tripod structure, the protein was no longer able to accumulate at midcell or cell poles. These findings favour a model where chemoreceptor localization in E. coli is driven by strong membrane curvature and association with the Tol-Pal complex. Importance Bacteria have exquisite mechanisms to sense and to adapt to the environment they live in. One such mechanism involves the chemotaxis signal transduction pathway, in which chemoreceptors specifically bind certain attracting or repelling molecules and transduce the signals to the cell. In different rod-shaped bacteria, these chemoreceptors localize specifically to cell poles. Here, we examined the polar localization of the aspartate chemoreceptor Tar in E. coli

Solid-state nanopore localization by controlled breakdown of selectively thinned membranes

NASA Astrophysics Data System (ADS)

Carlsen, Autumn T.; Briggs, Kyle; Hall, Adam R.; Tabard-Cossa, Vincent

2017-02-01

We demonstrate precise positioning of nanopores fabricated by controlled breakdown (CBD) on solid-state membranes by spatially varying the electric field strength with localized membrane thinning. We show 100 × 100 nm2 precision in standard SiN x membranes (30-100 nm thick) after selective thinning by as little as 25% with a helium ion beam. Control over nanopore position is achieved through the strong dependence of the electric field-driven CBD mechanism on membrane thickness. Confinement of pore formation to the thinned region of the membrane is confirmed by TEM imaging and by analysis of DNA translocations. These results enhance the functionality of CBD as a fabrication approach and enable the production of advanced nanopore devices for single-molecule sensing applications.

Hybrid membrane-microfluidic components using a novel ceramic MEMS technology

NASA Astrophysics Data System (ADS)

Lutz, Brent J.; Polyakov, Oleg; Rinaldo, Chris

2012-03-01

A novel hybrid nano/microfabrication technology has been employed to produce unique MEMS and microfluidic components that integrate nanoporous membranes. The components are made by micromachining a self-organized nanostructured ceramic material that is biocompatible and amenable to surface chemistry modification. Microfluidic structures, such as channels and wells, can be made with a precision of <2 microns. Thin-film membranes can be integrated into the bottom of these structures, featuring a wide range of possible thicknesses, from 100 micron to <50 nm. Additionally, these membranes may be non-porous or porous (with controllable pore sizes from 200 nm to <5 nm), for sophisticated size-based separations. With previous and current support from the NIH SBIR program, we have built several unique devices, and demonstrated improved separations, cell culturing, and imaging (optical and electron microscopy) versus standard products. Being ceramic, the material is much more robust to demanding environments (e.g. high and low temperatures and organic solvents), compared to polymer-based devices. Additionally, we have applied multiple surface modification techniques, including atomic layer deposition, to manipulate properties such as electrical conductivity. This microfabrication technology is highly scaleable, and thus can yield low-cost, reliable, disposable microcomponents and devices. Specific applications that can benefit from this technology includes cell culturing and assays, imaging by cryo-electron tomography, environmental sample processing, as well as many others.

Phosphatidylinositol kinase. A component of the chromaffin-granule membrane

PubMed Central

Phillips, John H.

1973-01-01

Phosphorylation of bovine chromaffin granules by ATP leads to the formation of diphosphoinositide in the granule membrane. Both phosphatidylinositol kinase and its substrate are components of this membrane, and triphosphoinositide is not formed under the conditions of the assay. The reaction is Mg2+-dependent and is stimulated by Mn2+ and F− ions. The initial reaction is rapid, with a broad pH profile and a `transition' temperature for its activation energy at 27°C. The apparent Km for ATP is 5μm. ATP, N-ethylmaleimide, Cu2+ ions and NaIO4 are inhibitory. The phospholipids of chromaffin-granule membranes have been analysed: 6.8% of the lipid P is found in phosphatidylinositol, and only 2–3% in phosphatidylserine. Comparison of the rate of phosphorylation of intact and lysed granules suggests that the sites for phosphorylation are on the outer (cytoplasmic) surface of the granules, and diphosphoinositide may therefore make an important contribution to the charge of the chromaffin granule in vivo. PMID:4360713

Cag3 Is a Novel Essential Component of the Helicobacter pylori Cag Type IV Secretion System Outer Membrane Subcomplex ▿ †

PubMed Central

Pinto-Santini, Delia M.; Salama, Nina R.

2009-01-01

Helicobacter pylori strains harboring the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI encodes a type IV secretion (T4S) system required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8). cag PAI genes sharing sequence similarity with T4S components from other bacteria are essential for Cag T4S function. Other cag PAI-encoded genes are also essential for Cag T4S, but lack of sequence-based or structural similarity with genes in existing databases has precluded a functional assignment for the encoded proteins. We have studied the role of one such protein, Cag3 (HP0522), in Cag T4S and determined Cag3 subcellular localization and protein interactions. Cag3 is membrane associated and copurifies with predicted inner and outer membrane Cag T4S components that are essential for Cag T4S as well as putative accessory factors. Coimmunoprecipitation and cross-linking experiments revealed specific interactions with HpVirB7 and CagM, suggesting Cag3 is a new component of the Cag T4S outer membrane subcomplex. Finally, lack of Cag3 lowers HpVirB7 steady-state levels, further indicating Cag3 makes a subcomplex with this protein. PMID:19801411

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.

A novel crystallization method for visualizing the membrane localization of potassium channels.

PubMed Central

Lopatin, A N; Makhina, E N; Nichols, C G

1998-01-01

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel. PMID:9591643

A novel crystallization method for visualizing the membrane localization of potassium channels.

PubMed

Lopatin, A N; Makhina, E N; Nichols, C G

1998-05-01

The high permeability of K+ channels to monovalent thallium (Tl+) ions and the low solubility of thallium bromide salt were used to develop a simple yet very sensitive approach to the study of membrane localization of potassium channels. K+ channels (Kir1.1, Kir2.1, Kir2.3, Kv2.1), were expressed in Xenopus oocytes and loaded with Br ions by microinjection. Oocytes were then exposed to extracellular thallium. Under conditions favoring influx of Tl+ ions (negative membrane potential under voltage clamp, or high concentration of extracellular Tl+), crystals of TlBr, visible under low-power microscopy, formed under the membrane in places of high density of K+ channels. Crystals were not formed in uninjected oocytes, but were formed in oocytes expressing as little as 5 microS K+ conductance. The number of observed crystals was much lower than the estimated number of functional channels. Based on the pattern of crystal formation, K+ channels appear to be expressed mostly around the point of cRNA injection when injected either into the animal or vegetal hemisphere. In addition to this pseudopolarized distribution of K+ channels due to localized microinjection of cRNA, a naturally polarized (animal/vegetal side) distribution of K+ channels was also frequently observed when K+ channel cRNA was injected at the equator. A second novel "agarose-hemiclamp" technique was developed to permit direct measurements of K+ currents from different hemispheres of oocytes under two-microelectrode voltage clamp. This technique, together with direct patch-clamping of patches of membrane in regions of high crystal density, confirmed that the localization of TlBr crystals corresponded to the localization of functional K+ channels and suggested a clustered organization of functional channels. With appropriate permeant ion/counterion pairs, this approach may be applicable to the visualization of the membrane distribution of any functional ion channel.

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

Palmitoylation of Sindbis Virus TF Protein Regulates Its Plasma Membrane Localization and Subsequent Incorporation into Virions.

PubMed

Ramsey, Jolene; Renzi, Emily C; Arnold, Randy J; Trinidad, Jonathan C; Mukhopadhyay, Suchetana

2017-02-01

Palmitoylation is a reversible, posttranslational modification that helps target proteins to cellular membranes. The alphavirus small membrane proteins 6K and TF have been reported to be palmitoylated and to positively regulate budding. 6K and TF are isoforms that are identical in their N termini but unique in their C termini due to a -1 ribosomal frameshift during translation. In this study, we used cysteine (Cys) mutants to test differential palmitoylation of the Sindbis virus 6K and TF proteins. We modularly mutated the five Cys residues in the identical N termini of 6K and TF, the four additional Cys residues in TF's unique C terminus, or all nine Cys residues in TF. Using these mutants, we determined that TF palmitoylation occurs primarily in the N terminus. In contrast, 6K is not palmitoylated, even on these shared residues. In the C-terminal Cys mutant, TF protein levels increase both in the cell and in the released virion compared to the wild type. In viruses with the N-terminal Cys residues mutated, TF is much less efficiently localized to the plasma membrane, and it is not incorporated into the virion. The three Cys mutants have minor defects in cell culture growth but a high incidence of abnormal particle morphologies compared to the wild-type virus as determined by transmission electron microscopy. We propose a model where the C terminus of TF modulates the palmitoylation of TF at the N terminus, and palmitoylated TF is preferentially trafficked to the plasma membrane for virus budding. Alphaviruses are a reemerging viral cause of arthritogenic disease. Recently, the small 6K and TF proteins of alphaviruses were shown to contribute to virulence in vivo Nevertheless, a clear understanding of the molecular mechanisms by which either protein acts to promote virus infection is missing. The TF protein is a component of budded virions, and optimal levels of TF correlate positively with wild-type-like particle morphology. In this study, we show that the

Fluorescent Labeling of the Nuclear Envelope by Localizing Green Fluorescent Protein on the Inner Nuclear Membrane.

PubMed

Taniyama, Toshiyuki; Tsuda, Natsumi; Sueda, Shinji

2018-06-15

The nuclear envelope (NE) is a double membrane that segregates nuclear components from the cytoplasm in eukaryotic cells. It is well-known that the NE undergoes a breakdown and reformation during mitosis in animal cells. However, the detailed mechanisms of the NE dynamics are not yet fully understood. Here, we propose a method for the fluorescent labeling of the NE in living cells, which enables the tracing of the NE dynamics during cell division under physiological conditions. In our method, labeling of the NE is accomplished by fixing green fluorescent protein carrying the nuclear localization signal on the inner nuclear membrane based on a unique biotinylation reaction from the archaeon Sulfolobus tokodaii. With this method, we observed HeLa cells during mitosis by confocal laser scanning microscopy and succeeded in clearly visualizing the difference in the timing of the formation of the NE and the nuclear lamina.

The correlation between biofilm biopolymer composition and membrane fouling in submerged membrane bioreactors.

PubMed

Luo, Jinxue; Zhang, Jinsong; Tan, Xiaohui; McDougald, Diane; Zhuang, Guoqiang; Fane, Anthony G; Kjelleberg, Staffan; Cohen, Yehuda; Rice, Scott A

2014-10-01

Biofouling, the combined effect of microorganism and biopolymer accumulation, significantly reduces the process efficiency of membrane bioreactors (MBRs). Here, four biofilm components, alpha-polysaccharides, beta-polysaccharides, proteins and microorganisms, were quantified in MBRs. The biomass of each component was positively correlated with the transmembrane pressure increase in MBRs. Proteins were the most abundant biopolymer in biofilms and showed the fastest rate of increase. The spatial distribution and co-localization analysis of the biofouling components indicated at least 60% of the extracellular polysaccharide (EPS) components were associated with the microbial cells when the transmembrane pressure (TMP) entered the jump phase, suggesting that the EPS components were either secreted by the biofilm cells or that the deposition of these components facilitated biofilm formation. It is suggested that biofilm formation and the accumulation of EPS are intrinsically coupled, resulting in biofouling and loss of system performance. Therefore, strategies that control biofilm formation on membranes may result in a significant improvement of MBR performance.

Demonstration of ion channel synthesis by isolated squid giant axon provides functional evidence for localized axonal membrane protein translation.

PubMed

Mathur, Chhavi; Johnson, Kory R; Tong, Brian A; Miranda, Pablo; Srikumar, Deepa; Basilio, Daniel; Latorre, Ramon; Bezanilla, Francisco; Holmgren, Miguel

2018-02-02

Local translation of membrane proteins in neuronal subcellular domains like soma, dendrites and axon termini is well-documented. In this study, we isolated the electrical signaling unit of an axon by dissecting giant axons from mature squids (Dosidicus gigas). Axoplasm extracted from these axons was found to contain ribosomal RNAs, ~8000 messenger RNA species, many encoding the translation machinery, membrane proteins, translocon and signal recognition particle (SRP) subunits, endomembrane-associated proteins, and unprecedented proportions of SRP RNA (~68% identical to human homolog). While these components support endoplasmic reticulum-dependent protein synthesis, functional assessment of a newly synthesized membrane protein in axolemma of an isolated axon is technically challenging. Ion channels are ideal proteins for this purpose because their functional dynamics can be directly evaluated by applying voltage clamp across the axon membrane. We delivered in vitro transcribed RNA encoding native or Drosophila voltage-activated Shaker K V channel into excised squid giant axons. We found that total K + currents increased in both cases; with added inactivation kinetics on those axons injected with RNA encoding the Shaker channel. These results provide unambiguous evidence that isolated axons can exhibit de novo synthesis, assembly and membrane incorporation of fully functional oligomeric membrane proteins.

DNA molecules on periodically microstructured lipid membranes: Localization and coil stretching

NASA Astrophysics Data System (ADS)

Hochrein, Marion B.; Leierseder, Judith A.; Golubović, Leonardo; Rädler, Joachim O.

2007-02-01

We explore large scale conformations of DNA molecules adsorbed on curved surfaces. For that purpose, we investigate the behavior of DNA adsorbed on periodically shaped cationic lipid membranes. These unique membrane morphologies are supported on grooved, one-dimensionally periodic microstructured surfaces. Strikingly, we find that these periodically structured membranes are capable to stretch DNA coils. We elucidate this phenomenon in terms of surface curvature dependent potential energy attained by the adsorbed DNA molecules. Due to it, DNA molecules undergo a localization transition causing them to stretch by binding to highly curved sections (edges) of the supported membranes. This effect provides a new venue for controlling conformations of semiflexible polymers such as DNA by employing their interactions with specially designed biocompatible surfaces. We report the first experimental observation of semiflexible polymers unbinding transition in which DNA molecules unbind from one-dimensional manifolds (edges) while remaining bound to two-dimensional manifolds (cationic membranes).

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.

Transcriptomic insights into citrus segment membrane's cell wall components relating to fruit sensory texture.

PubMed

Wang, Xun; Lin, Lijin; Tang, Yi; Xia, Hui; Zhang, Xiancong; Yue, Maolan; Qiu, Xia; Xu, Ke; Wang, Zhihui

2018-04-23

During fresh fruit consumption, sensory texture is one factor that affects the organoleptic qualities. Chemical components of plant cell walls, including pectin, cellulose, hemicellulose and lignin, play central roles in determining the textural qualities. To explore the genes and regulatory pathways involved in fresh citrus' perceived sensory texture, we performed mRNA-seq analyses of the segment membranes of two citrus cultivars, Shiranui and Kiyomi, with different organoleptic textures. Segment membranes were sampled at two developmental stages of citrus fruit, the beginning and end of the expansion period. More than 3000 differentially expressed genes were identified. The gene ontology analysis revealed that more categories were significantly enriched in 'Shiranui' than in 'Kiyomi' at both developmental stages. In total, 108 significantly enriched pathways were obtained, with most belonging to metabolism. A detailed transcriptomic analysis revealed potential critical genes involved in the metabolism of cell wall structures, for example, GAUT4 in pectin synthesis, CESA1, 3 and 6, and SUS4 in cellulose synthesis, CSLC5, XXT1 and XXT2 in hemicellulose synthesis, and CSE in lignin synthesis. Low levels, or no expression, of genes involved in cellulose and hemicellulose, such as CESA4, CESA7, CESA8, IRX9 and IRX14, confirmed that secondary cell walls were negligible or absent in citrus segment membranes. A chemical component analysis of the segment membranes from mature fruit revealed that the pectin, cellulose and lignin contents, and the segment membrane's weight (% of segment) were greater in 'Kiyomi'. Organoleptic quality of citrus is easily overlooked. It is mainly determined by sensory texture perceived in citrus segment membrane properties. We performed mRNA-seq analyses of citrus segment membranes to explore the genes and regulatory pathways involved in fresh citrus' perceived sensory texture. Transcriptomic data showed high repeatability between two

ERAD components in organisms with complex red plastids suggest recruitment of a preexisting protein transport pathway for the periplastid membrane.

PubMed

Felsner, Gregor; Sommer, Maik S; Gruenheit, Nicole; Hempel, Franziska; Moog, Daniel; Zauner, Stefan; Martin, William; Maier, Uwe G

2011-01-01

The plastids of cryptophytes, haptophytes, and heterokontophytes (stramenopiles) (together once known as chromists) are surrounded by four membranes, reflecting the origin of these plastids through secondary endosymbiosis. They share this trait with apicomplexans, which are alveolates, the plastids of which have been suggested to stem from the same secondary symbiotic event and therefore form a phylogenetic clade, the chromalveolates. The chromists are quantitatively the most important eukaryotic contributors to primary production in marine ecosystems. The mechanisms of protein import across their four plastid membranes are still poorly understood. Components of an endoplasmic reticulum-associated degradation (ERAD) machinery in cryptophytes, partially encoded by the reduced genome of the secondary symbiont (the nucleomorph), are implicated in protein transport across the second outermost plastid membrane. Here, we show that the haptophyte Emiliania huxleyi, like cryptophytes, stramenopiles, and apicomplexans, possesses a nuclear-encoded symbiont-specific ERAD machinery (SELMA, symbiont-specific ERAD-like machinery) in addition to the host ERAD system, with targeting signals that are able to direct green fluorescent protein or yellow fluorescent protein to the predicted cellular localization in transformed cells of the stramenopile Phaeodactylum tricornutum. Phylogenies of the duplicated ERAD factors reveal that all SELMA components trace back to a red algal origin. In contrast, the host copies of cryptophytes and haptophytes associate with the green lineage to the exclusion of stramenopiles and alveolates. Although all chromalveolates with four membrane-bound plastids possess the SELMA system, this has apparently not arisen in a single endosymbiotic event. Thus, our data do not support the chromalveolate hypothesis.

Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating PI3K membrane localization in Dictyostelium

PubMed Central

Sun, Tong; Kim, Bohye; Kim, Lou W.

2013-01-01

Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved in diverse cellular activities such as metabolism, differentiation, and morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3 affects PI3K membrane localization, of which mechanism has remained to be fully understood in Dictyostelium. The membrane localization domain (LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased membrane localization in gsk3− cells compared to the wild type cells. Furthermore, multiple GSK3-phosphorylation consensus sites exist in PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced transient membrane localization of PI3K1-LD in gsk3− cells. Serine to alanine substitution mutants of PI3K1-LD, in contrast, displayed constitutive membrane localization in wild type cells. Biochemical analysis revealed that GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during the course of cAMP stimulation. Together, these data suggest that GSK3 dependent serine phosphorylation is a prerequisite for chemoattractant cAMP induced PI3K membrane localization. PMID:24102085

Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling.

PubMed

Elías-Wolff, Federico; Lindén, Martin; Lyubartsev, Alexander P; Brandt, Erik G

2018-03-13

Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane-associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase.

PubMed

Haruta, Miyoshi; Tan, Li Xuan; Bushey, Daniel B; Swanson, Sarah J; Sussman, Michael R

2018-01-01

A P-type H + -ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis ( Arabidopsis thaliana ) plant expressing H + -ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H + secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H + -ATPase. © 2018 American Society of Plant Biologists. All Rights Reserved.

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

PubMed

Morigaki, Kenichi; Tanimoto, Yasushi

2018-03-14

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

Biocatalytic Stimuli-Responsive Asymmetric Triblock Terpolymer Membranes for Localized Permeability Gating.

PubMed

Poole, Jennifer L; Donahue, Scott; Wilson, David; Li, Yuk Mun; Zhang, Qi; Gu, Yibei; Ferebee, Rachel; Lu, Zhao; Dorin, Rachel Mika; Hancock, Lawrence F; Takiff, Larry; Hakem, Ilhem F; Bockstaller, Michael R; Wiesner, Ulrich; Walker, Jeremy

2017-10-01

The functionalization with phosphotriesterase of poly(isoprene-b-styrene-b-4-vinylpyridine)-based nanoporous membranes fabricated by self-assembly and nonsolvent induced phase separation (SNIPS) is shown to enable dynamically responsive membranes capable of substrate-specific and localized gating response. Integration of the SNIPS process with macroporous nylon support layers yields mechanically robust textile-type films with high moisture vapor transport rates that display rapid and local order-of-magnitude modulation of permeability. The simplicity of the fabrication process that is compatible with large-area fabrication along with the versatility and efficacy of enzyme reactivity offers intriguing opportunities for engineered biomimetic materials that are tailored to respond to a complex range of external parameters, providing sensing, protection, and remediation capabilities. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sperm flagella protein components: Human meichroacidin constructed by the membrane occupation and recognition nexus motif

PubMed Central

MATSUOKA, YASUHIRO; NISHIMURA, HIROMI; NUMAZAWA, KAHORI; TSUCHIDA, JUNJI; MIYAGAWA, YASUSHI; TSUJIMURA, AKIRA; MATSUMIYA, KIYOMI; OKUYAMA, AKIHIKO; NISHIMUNE, YOSHITAKE

2005-01-01

Background and Aims:   In a previous study, the authors of the present study cloned mouse meichroacidin (MCA), which is expressed in stages of spermatogenesis from pachytene spermatocytes through round spermatid germ cells. MCA protein contains the membrane occupation and recognition nexus (MORN) motif and localizes to a male meiotic metaphase chromosome. Recently, a MCA homolog of carp (Cyprinus carpio), MORN motif‐containing sperm‐specific axonemal protein (MSAP), was reportedly identified and localized in sperm flagella. Present knowledge of human spermiogenesis requires the identification of proteins in human sperm. The present study identified the human orthologue of MCA. Methods:   Colony hybridization using a human testis plasmid cDNA library was carried out to clone human MCA (h‐MCA) cDNA. Northern blot, Western blot, and immunohistochemical analyses were carried out. Results:   h‐MCA was found to be specifically expressed in the testes. The h‐MCA amino acid sequence shared 79.8% identity with mouse MCA and contained MORN motifs. h‐MCA localized in the sperm flagellum and basal body, as does MSAP in carp. Conclusion:   Expression and localization analyses showed that h‐MCA is a component of the sperm flagellum and basal body and might play an important role in the development of the sperm flagellum in humans. (Reprod Med Biol 2005; 4: 213–219) PMID:29699225

Problem-Solving Test: Submitochondrial Localization of Proteins

ERIC Educational Resources Information Center

Szeberenyi, Jozsef

2011-01-01

Mitochondria are surrounded by two membranes (outer and inner mitochondrial membrane) that separate two mitochondrial compartments (intermembrane space and matrix). Hundreds of proteins are distributed among these submitochondrial components. A simple biochemical/immunological procedure is described in this test to determine the localization of…

Measuring Local Viscosities near Plasma Membranes of Living Cells with Photonic Force Microscopy

PubMed Central

Jünger, Felix; Kohler, Felix; Meinel, Andreas; Meyer, Tim; Nitschke, Roland; Erhard, Birgit; Rohrbach, Alexander

2015-01-01

The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle’s hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 μm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 μs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions. PMID:26331245

Lattice Independent Component Analysis for Mobile Robot Localization

NASA Astrophysics Data System (ADS)

Villaverde, Ivan; Fernandez-Gauna, Borja; Zulueta, Ekaitz

This paper introduces an approach to appearance based mobile robot localization using Lattice Independent Component Analysis (LICA). The Endmember Induction Heuristic Algorithm (EIHA) is used to select a set of Strong Lattice Independent (SLI) vectors, which can be assumed to be Affine Independent, and therefore candidates to be the endmembers of the data. Selected endmembers are used to compute the linear unmixing of the robot's acquired images. The resulting mixing coefficients are used as feature vectors for view recognition through classification. We show on a sample path experiment that our approach can recognise the localization of the robot and we compare the results with the Independent Component Analysis (ICA).

Localized Patch Clamping of Plasma Membrane of a Polarized Plant Cell 1

PubMed Central

Taylor, Alison R.; Brownlee, Colin

1992-01-01

We used an ultraviolet laser to rupture a small region of cell wall of a polarized Fucus spiralis rhizoid cell and gained localized access to the plasma membrane at the growing apex. Careful control of cell turgor enabled a small portion of plasma membrane-bound cytoplasm to be exposed. Gigaohm seals allowing single-channel recordings were obtained with a high success rate using this method with conventional patch clamp techniques. ImagesFigure 1 PMID:16669092

[Optimization of labeling and localizing bacterial membrane and nucleus with FM4-64 and Hoechst dyes].

PubMed

Wang, Jing; Han, Yanping; Yang, Ruifu; Zhao, Xingxu

2015-08-04

To observe cell membrane and nucleus in bacteria for subcellular localization. FM4-64 and Hoechst were dyed that can label cell membrane and nucleus, respectively. Both dyes were used to co-stain the membranes and nucleus of eight bacterial strains ( Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Yersinia pestis, Legionella pneumonia, Vibrio cholerae and Bacillus anthracis). E. coli was dyed with different dye concentrations and times and then observed by confocal fluorescence microscopic imaging. Fluorescence intensity of cell membrane and nucleus is affected by dye concentrations and times. The optimal conditions were determined as follows: staining cell membrane with 20 μg/mL FM4-64 for 1 min and cell nucleus with 20 μg/mL Hoechst for 20 min. Gram-negative bacteria were dyed better than gram-positive bacteria with FM4-64dye. FM4-64 and Hoechst can be used to stain membrane and nucleus in different types of bacteria. Co-staining bacterial membrane and nucleus provides the reference to observe cell structure in prokaryotes for studying subcellular localization.

X11/Mint Genes Control Polarized Localization of Axonal Membrane Proteins in Vivo

PubMed Central

Gross, Garrett G.; Lone, G. Mohiddin; Leung, Lok Kwan; Hartenstein, Volker

2013-01-01

Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body in Drosophila, a brain structure involved in learning and memory. Axonal localization of APP is mediated by an endocytic motif, and loss of X11/Mint results in a dramatic increase in cell-surface levels of APPL, especially on dendrites. Mutations in genes required for endocytosis show similar mislocalization of these proteins to dendrites, and strongly enhance defects seen in X11/Mint mutants. These results suggest that X11/Mint-dependent endocytosis in dendrites may serve to promote the axonal localization of membrane proteins. Since X11/Mint binds to APP, and abnormal trafficking of APP contributes to Alzheimer's disease, deregulation of X11/Mint may be important for Alzheimer's disease pathogenesis. PMID:23658195

Membrane-Based Characterization of a Gas Component — A Transient Sensor Theory

PubMed Central

Lazik, Detlef

2014-01-01

Based on a multi-gas solution-diffusion problem for a dense symmetrical membrane this paper presents a transient theory of a planar, membrane-based sensor cell for measuring gas from both initial conditions: dynamic and thermodynamic equilibrium. Using this theory, the ranges for which previously developed, simpler approaches are valid will be discussed; these approaches are of vital interest for membrane-based gas sensor applications. Finally, a new theoretical approach is introduced to identify varying gas components by arranging sensor cell pairs resulting in a concentration independent gas-specific critical time. Literature data for the N2, O2, Ar, CH4, CO2, H2 and C4H10 diffusion coefficients and solubilities for a polydimethylsiloxane membrane were used to simulate gas specific sensor responses. The results demonstrate the influence of (i) the operational mode; (ii) sensor geometry and (iii) gas matrices (air, Ar) on that critical time. Based on the developed theory the case-specific suitable membrane materials can be determined and both operation and design options for these sensors can be optimized for individual applications. The results of mixing experiments for different gases (O2, CO2) in a gas matrix of air confirmed the theoretical predictions. PMID:24608004

Establishment of A431 cell membrane chromatography-RPLC method for screening target components from Radix Caulophylli.

PubMed

Hou, Xiaofang; Wang, Sicen; Hou, Jingjing; He, Langchong

2011-03-01

We describe here an analytical method of A431 cell membrane chromatography (A431/CMC) (CMC, cell membrane chromatography) combined with RPLC for recognition, separation, and identification of target components from traditional Chinese medicines (TCMs) Radix Caulophylli. The A431 cells with high expressed epidermal growth factor receptor (EGFR) were used to prepare the stationary phase in the CMC model. Retention fractions on the A431-CMC model were collected using an automated fraction collection and injection module (FC/I). Each fraction was analyzed by RPLC under the optimized conditions. Gefitinib and erlotinib were used as standard compounds to investigate the suitability and reliability of the A431 cell membrane chromatography-RPLC method prior to screening target component from Radix Caulophylli total alkaloids. The results indicated that caulophine and taspine were the target component acting on the epidermal growth factor receptor. This method could be an efficient way in drug discovery using natural medicinal herbs as a source of novel compounds. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission

PubMed Central

van der Hoeven, Dharini; Cho, Kwang-jin; Ma, Xiaoping; Chigurupati, Sravanthi; Parton, Robert G.

2013-01-01

Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics. PMID:23129805

Influence of Global and Local Membrane Curvature on Mechanosensitive Ion Channels: A Finite Element Approach

PubMed Central

Bavi, Omid; Cox, Charles D.; Vossoughi, Manouchehr; Naghdabadi, Reza; Jamali, Yousef; Martinac, Boris

2016-01-01

Mechanosensitive (MS) channels are ubiquitous molecular force sensors that respond to a number of different mechanical stimuli including tensile, compressive and shear stress. MS channels are also proposed to be molecular curvature sensors gating in response to bending in their local environment. One of the main mechanisms to functionally study these channels is the patch clamp technique. However, the patch of membrane surveyed using this methodology is far from physiological. Here we use continuum mechanics to probe the question of how curvature, in a standard patch clamp experiment, at different length scales (global and local) affects a model MS channel. Firstly, to increase the accuracy of the Laplace’s equation in tension estimation in a patch membrane and to be able to more precisely describe the transient phenomena happening during patch clamping, we propose a modified Laplace’s equation. Most importantly, we unambiguously show that the global curvature of a patch, which is visible under the microscope during patch clamp experiments, is of negligible energetic consequence for activation of an MS channel in a model membrane. However, the local curvature (RL < 50) and the direction of bending are able to cause considerable changes in the stress distribution through the thickness of the membrane. Not only does local bending, in the order of physiologically relevant curvatures, cause a substantial change in the pressure profile but it also significantly modifies the stress distribution in response to force application. Understanding these stress variations in regions of high local bending is essential for a complete understanding of the effects of curvature on MS channels. PMID:26861405

Modeling of membrane processes for air revitalization and water recovery

NASA Technical Reports Server (NTRS)

Lange, Kevin E.; Foerg, Sandra L.; Dall-Bauman, Liese A.

1992-01-01

Gas-separation and reverse-osmosis membrane models are being developed in conjunction with membrane testing at NASA JSC. The completed gas-separation membrane model extracts effective component permeabilities from multicomponent test data, and predicts the effects of flow configuration, operating conditions, and membrane dimensions on module performance. Variable feed- and permeate-side pressures are considered. The model has been applied to test data for hollow-fiber membrane modules with simulated cabin-air feeds. Results are presented for a membrane designed for air drying applications. Extracted permeabilities are used to predict the effect of operating conditions on water enrichment in the permeate. A first-order reverse-osmosis model has been applied to test data for spiral wound membrane modules with a simulated hygiene water feed. The model estimates an effective local component rejection coefficient under pseudosteady-state conditions. Results are used to define requirements for a detailed reverse-osmosis model.

ADP-ribosylation of membrane components by pertussis and cholera toxin

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

Ribeiro-Neto, F.A.P.; Mattera, F.; Hildebrandt, J.D.

1985-01-01

Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (N/sub s/) and inhibitory (N/sub i/) regulatory components of adenylyl cyclase. Cholera toxin acts on N/sub s/ by ADP-ribosylating its ..cap alpha../sub s/ subunit; pertussis toxin acts on N/sub i/ by ADP-ribosylating its ..cap alpha..; subunit. By using (/sup 32/P)NAD/sup +/ and determining the transfer of its (/sup 32/P)ADP-ribose moiety to membrane components, it is possible to obtain information on N/sub s/ and N/sub i/. A set of protocols is presented that can be used to study simultaneously and comparatively the susceptibility of N/submore » s/ and N/sub i/ to be ADP-ribosylated by cholera and pertussis toxin.« less

Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes.

PubMed

Casbon, Amy-Jo; Allen, Lee-Ann H; Dunn, Kenneth W; Dinauer, Mary C

2009-02-15

Flavocytochrome b(558), the catalytic core of the phagocytic NADPH oxidase, mediates the transfer of electrons from NADPH to molecular oxygen to generate superoxide for host defense. Flavocytochrome b is a membrane heterodimer consisting of a large subunit gp91(phox) (NOX2) and a smaller subunit, p22(phox). Although in neutrophils flavocytochrome b has been shown to localize to the plasma membrane and specific granules, little is known about its distribution in macrophages. Using immunofluorescent staining and live cell imaging of fluorescently tagged gp91(phox) and p22(phox), we demonstrate in a Chinese hamster ovary cell model system and in RAW 264.7 and primary murine bone marrow-derived macrophages that flavocytochrome b is found in the Rab11-positive recycling endocytic compartment, as well as in Rab5-positive early endosomes and plasma membrane. Additionally, we show that unassembled p22(phox) and gp91(phox) subunits localize to the endoplasmic reticulum, which redistribute to the cell surface and endosomal compartments following heterodimer formation. These studies show for the first time that flavocytochrome b localizes to intracellular compartments in macrophages that recycle to the plasma membrane, which may act as a reservoir to deliver flavocytochrome b to the cell surface and phagosome membranes.

Steric exclusion and protein conformation determine the localization of plasma membrane transporters.

PubMed

Bianchi, Frans; Syga, Łukasz; Moiset, Gemma; Spakman, Dian; Schavemaker, Paul E; Punter, Christiaan M; Seinen, Anne-Bart; van Oijen, Antoine M; Robinson, Andrew; Poolman, Bert

2018-02-05

The plasma membrane (PM) of Saccharomyces cerevisiae contains membrane compartments, MCC/eisosomes and MCPs, named after the protein residents Can1 and Pma1, respectively. Using high-resolution fluorescence microscopy techniques we show that Can1 and the homologous transporter Lyp1 are able to diffuse into the MCC/eisosomes, where a limited number of proteins are conditionally trapped at the (outer) edge of the compartment. Upon addition of substrate, the immobilized proteins diffuse away from the MCC/eisosomes, presumably after taking a different conformation in the substrate-bound state. Our data indicate that the mobile fraction of all integral plasma membrane proteins tested shows extremely slow Brownian diffusion through most of the PM. We also show that proteins with large cytoplasmic domains, such as Pma1 and synthetic chimera of Can1 and Lyp1, are excluded from the MCC/eisosomes. We hypothesize that the distinct localization patterns found for these integral membrane proteins in S. cerevisiae arises from a combination of slow lateral diffusion, steric exclusion, and conditional trapping in membrane compartments.

Dual effect of local anesthetics on the function of excitable rod outer segment disk membrane

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

Mashimo, T.; Abe, K.; Yoshiya, I.

1986-04-01

The effects of local anesthetics and a divalent cation, Ca2+, on the function of rhodopsin were estimated from the measurements of light-induced proton uptake. The light-induced proton uptake by rhodopsin in the rod outer segment disk membrane was enhanced at lower pH (4) but depressed at higher pHs (6 to 8) by the tertiary amine local anesthetics lidocaine, bupivacaine, tetracaine, and dibucaine. The order of local anesthetic-induced depression of the proton uptake followed that of their clinical anesthetic potencies. The depression of the proton uptake versus the concentration of the uncharged form of local anesthetic nearly describes the same curvemore » for small and large dose of added anesthetic. Furthermore, a neutral local anesthetic, benzocaine, depressed the proton uptake at all pHs between 4 and 7. These results indicate that the depression of the proton uptake is due to the effect of only the uncharged form. It is hypothesized that the uncharged form of local anesthetics interacts hydrophobically with the rhodopsin in the disk membrane. The dual effect of local anesthetics on the proton uptake, on the other hand, suggests that the activation of the function of rhodopsin may be caused by the charged form. There was no significant change in the light-induced proton uptake by rhodopsin when 1 mM of Ca2+ was introduced into the disk membrane at varying pHs in the absence or presence of local anesthetics. This fact indicates that Ca2+ ion does not influence the diprotonating process of metarhodopsin; neither does it interfere with the local anesthetic-induced changes in the rhodopsin molecule.« less

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane.

PubMed

Stone, Matthew B; Shelby, Sarah A; Veatch, Sarah L

2017-06-14

Lipids and the membranes they form are fundamental building blocks of cellular life, and their geometry and chemical properties distinguish membranes from other cellular environments. Collective processes occurring within membranes strongly impact cellular behavior and biochemistry, and understanding these processes presents unique challenges due to the often complex and myriad interactions between membrane components. Super-resolution microscopy offers a significant gain in resolution over traditional optical microscopy, enabling the localization of individual molecules even in densely labeled samples and in cellular and tissue environments. These microscopy techniques have been used to examine the organization and dynamics of plasma membrane components, providing insight into the fundamental interactions that determine membrane functions. Here, we broadly introduce the structure and organization of the mammalian plasma membrane and review recent applications of super-resolution microscopy to the study of membranes. We then highlight some inherent challenges faced when using super-resolution microscopy to study membranes, and we discuss recent technical advancements that promise further improvements to super-resolution microscopy and its application to the plasma membrane.

Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase1[OPEN

PubMed Central

Tan, Li Xuan; Bushey, Daniel B.; Swanson, Sarah J.

2018-01-01

A P-type H+-ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis (Arabidopsis thaliana) plant expressing H+-ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H+ secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H+-ATPase. PMID:29042459

Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

PubMed

Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

2016-05-01

Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, <0.9). For the first time, we have demonstrated that mitochondrial MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β. © FASEB.

Progesterone receptor membrane component-1 regulates hepcidin biosynthesis

PubMed Central

Li, Xiang; Rhee, David K.; Malhotra, Rajeev; Mayeur, Claire; Hurst, Liam A.; Ager, Emily; Shelton, Georgia; Kramer, Yael; McCulloh, David; Keefe, David; Bloch, Kenneth D.; Bloch, Donald B.; Peterson, Randall T.

2015-01-01

Iron homeostasis is tightly regulated by the membrane iron exporter ferroportin and its regulatory peptide hormone hepcidin. The hepcidin/ferroportin axis is considered a promising therapeutic target for the treatment of diseases of iron overload or deficiency. Here, we conducted a chemical screen in zebrafish to identify small molecules that decrease ferroportin protein levels. The chemical screen led to the identification of 3 steroid molecules, epitiostanol, progesterone, and mifepristone, which decrease ferroportin levels by increasing the biosynthesis of hepcidin. These hepcidin-inducing steroids (HISs) did not activate known hepcidin-inducing pathways, including the BMP and JAK/STAT3 pathways. Progesterone receptor membrane component-1 (PGRMC1) was required for HIS-dependent increases in hepcidin biosynthesis, as PGRMC1 depletion in cultured hepatoma cells and zebrafish blocked the ability of HISs to increase hepcidin mRNA levels. Neutralizing antibodies directed against PGRMC1 attenuated the ability of HISs to induce hepcidin gene expression. Inhibiting the kinases of the SRC family, which are downstream of PGRMC1, blocked the ability of HISs to increase hepcidin mRNA levels. Furthermore, HIS treatment increased hepcidin biosynthesis in mice and humans. Together, these data indicate that PGRMC1 regulates hepcidin gene expression through an evolutionarily conserved mechanism. These studies have identified drug candidates and potential therapeutic targets for the treatment of diseases of abnormal iron metabolism. PMID:26657863

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

PubMed Central

Dyrda, Agnieszka; Cytlak, Urszula; Ciuraszkiewicz, Anna; Lipinska, Agnieszka; Cueff, Anne; Bouyer, Guillaume; Egée, Stéphane; Bennekou, Poul; Lew, Virgilio L.; Thomas, Serge L. Y.

2010-01-01

Background The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. Methodology/Principal Findings The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K+ and Cl− currents were strictly dependent on the presence of Ca2+. The Ca2+-dependent currents were transient, with typical decay half-times of about 5–10 min, suggesting the spontaneous inactivation of a stretch-activated Ca2+ permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca2+ permeability pathway leading to increased [Ca2+]i, secondary activation of Ca2+-sensitive K+ channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. Conclusions/Significance The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca2+-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca2+ content

Local membrane deformations activate Ca2+-dependent K+ and anionic currents in intact human red blood cells.

PubMed

Dyrda, Agnieszka; Cytlak, Urszula; Ciuraszkiewicz, Anna; Lipinska, Agnieszka; Cueff, Anne; Bouyer, Guillaume; Egée, Stéphane; Bennekou, Poul; Lew, Virgilio L; Thomas, Serge L Y

2010-02-26

The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K(+) and Cl(-) currents were strictly dependent on the presence of Ca(2+). The Ca(2+)-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+) permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca(2+) permeability pathway leading to increased [Ca(2+)](i), secondary activation of Ca(2+)-sensitive K(+) channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias

MEMBRANE FILTER PROCEDURE FOR ENUMERATING THE COMPONENT GENERA OF THE COLIFORM GROUP IN SEAWATER

EPA Science Inventory

A facile, quantitative, membrane filter procedure (mC) for defining the distribution of coliform populations in seawater according to the component genera was developed. The procedure, which utilizes a series of in situ substrate tests to obviate the picking of colonies for ident...

Effect of Cholesterol on the Structure of a Five-Component Mitochondria-Like Phospholipid Membrane

PubMed Central

Cathcart, Kelly; Patel, Amit; Dies, Hannah; Rheinstädter, Maikel C.; Fradin, Cécile

2015-01-01

Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails. This particular system was chosen because elevated cholesterol contents in mitochondrial membranes have been linked to a breaking down of Bax-mediated membrane permeabilization and resistance to cancer treatments. High resolution electron density profiles were determined by X-ray reflectivity, while the area per phospholipid chain, Apc, and the chain order parameter, SX-ray, were determined by wide-angle X-ray scattering (WAXS). We show that chain order increases upon the addition of cholesterol, resulting in both a thickening of the lipid bilayer and a reduction in the average surface area per phospholipid chain. This effect, well known as cholesterol’s condensation effect, is similar, but not as pronounced as for single-component phospholipid membranes. We conclude by discussing the relevance of these findings for the insertion of the pro-apoptotic protein Bax in mitochondrial membranes with elevated cholesterol content. PMID:26529029

Effect of Cholesterol on the Structure of a Five-Component Mitochondria-Like Phospholipid Membrane.

PubMed

Cathcart, Kelly; Patel, Amit; Dies, Hannah; Rheinstädter, Maikel C; Fradin, Cécile

2015-10-30

Cellular membranes have a complex phospholipid composition that varies greatly depending on the organism, cell type and function. In spite of this complexity, most structural data available for phospholipid bilayers concern model systems containing only one or two different phospholipids. Here, we examine the effect of cholesterol on the structure of a complex membrane reflecting the lipid composition of mitochondrial membranes, with five different types of headgroups (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS) and cardiolipin (CL)) and a variety of hydrocarbon tails. This particular system was chosen because elevated cholesterol contents in mitochondrial membranes have been linked to a breaking down of Bax-mediated membrane permeabilization and resistance to cancer treatments. High resolution electron density profiles were determined by X-ray reflectivity, while the area per phospholipid chain, Apc, and the chain order parameter, SX-ray, were determined by wide-angle X-ray scattering (WAXS). We show that chain order increases upon the addition of cholesterol, resulting in both a thickening of the lipid bilayer and a reduction in the average surface area per phospholipid chain. This effect, well known as cholesterol's condensation effect, is similar, but not as pronounced as for single-component phospholipid membranes. We conclude by discussing the relevance of these findings for the insertion of the pro-apoptotic protein Bax in mitochondrial membranes with elevated cholesterol content.

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.

Screening anti-allergic components of Astragali Radix using LAD2 cell membrane chromatography coupled online with UHPLC-ESI-MS/MS method.

PubMed

Lv, Yanni; Sun, Yueming; Fu, Jia; Kong, Liyun; Han, Shengli

2017-02-01

Huangqi (Astragali Radix), a traditional Chinese herb, is widely used in clinical therapy in China. In addition, an anti-allergic effect of constituents in Huangqi has been reported in the scientific literature. In the present study, cell membrane chromatography coupled online with UHPLC-ESI-MS/MS method was developed to screen, analyze and identify the anti-allergic components of Huangqi. The Laboratory of Allergic Disease 2 (LAD2) cell was used to establish cell membrane chromatography, which was combined with UHPLC-ESI-MS/MS. The coupled system was then used to screen anti-allergic components from Huangqi. Effects of active components were verified by histamine release assay. A component retained on the LAD2 cell membrane chromatography was identified as formononetin. Bioactivity of formononetin was investigated by histamine release assay in LAD2 cells, and it was found that formononetin could inhibit histamine release in a dose-dependent manner from 1 to 100 μm. The LAD2 cell membrane chromatography online with UHPLC-ESI-MS/MS method is an effective technique for screening the anti-allergic components of Huangqi. Copyright © 2016 John Wiley & Sons, Ltd.

Local Area Water Removal Analysis of a Proton Exchange Membrane Fuel Cell under Gas Purge Conditions

PubMed Central

Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

2012-01-01

In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold. PMID:22368495

Local area water removal analysis of a proton exchange membrane fuel cell under gas purge conditions.

PubMed

Lee, Chi-Yuan; Lee, Yu-Ming; Lee, Shuo-Jen

2012-01-01

In this study, local area water content distribution under various gas purging conditions are experimentally analyzed for the first time. The local high frequency resistance (HFR) is measured using novel micro sensors. The results reveal that the liquid water removal rate in a membrane electrode assembly (MEA) is non-uniform. In the under-the-channel area, the removal of liquid water is governed by both convective and diffusive flux of the through-plane drying. Thus, almost all of the liquid water is removed within 30 s of purging with gas. However, liquid water that is stored in the under-the-rib area is not easy to remove during 1 min of gas purging. Therefore, the re-hydration of the membrane by internal diffusive flux is faster than that in the under-the-channel area. Consequently, local fuel starvation and membrane degradation can degrade the performance of a fuel cell that is started from cold.

The RxLR effector Avh241 from Phytophthora sojae requires plasma membrane localization to induce plant cell death.

PubMed

Yu, Xiaoli; Tang, Junli; Wang, Qunqing; Ye, Wenwu; Tao, Kai; Duan, Shuyi; Lu, Chenchen; Yang, Xinyu; Dong, Suomeng; Zheng, Xiaobo; Wang, Yuanchao

2012-10-01

• The Phytophthora sojae genome encodes hundreds of RxLR effectors predicted to manipulate various plant defense responses, but the molecular mechanisms involved are largely unknown. Here we have characterized in detail the P. sojae RxLR effector Avh241. • To determine the function and localization of Avh241, we transiently expressed it on different plants. Silencing of Avh241 in P. sojae, we determined its virulence during infection. Through the assay of promoting infection by Phytophthora capsici to Nicotiana benthamiana, we further confirmed this virulence role. • Avh241 induced cell death in several different plants and localized to the plant plasma membrane. An N-terminal motif within Avh241 was important for membrane localization and cell death-inducing activity. Two mitogen-activated protein kinases, NbMEK2 and NbWIPK, were required for the cell death triggered by Avh241 in N. benthamiana. Avh241 was important for the pathogen's full virulence on soybean. Avh241 could also promote infection by P. capsici and the membrane localization motif was not required to promote infection. • This work suggests that Avh241 interacts with the plant immune system via at least two different mechanisms, one recognized by plants dependent on subcellular localization and one promoting infection independent on membrane localization. © 2012 The Authors. New Phytologist © 2012 New Phytologist Trust.

Localized waves in three-component coupled nonlinear Schrödinger equation

NASA Astrophysics Data System (ADS)

Xu, Tao; Chen, Yong

2016-09-01

We study the generalized Darboux transformation to the three-component coupled nonlinear Schrödinger equation. First- and second-order localized waves are obtained by this technique. In first-order localized wave, we get the interactional solutions between first-order rogue wave and one-dark, one-bright soliton respectively. Meanwhile, the interactional solutions between one-breather and first-order rogue wave are also given. In second-order localized wave, one-dark-one-bright soliton together with second-order rogue wave is presented in the first component, and two-bright soliton together with second-order rogue wave are gained respectively in the other two components. Besides, we observe second-order rogue wave together with one-breather in three components. Moreover, by increasing the absolute values of two free parameters, the nonlinear waves merge with each other distinctly. These results further reveal the interesting dynamic structures of localized waves in the three-component coupled system. Project supported by the Global Change Research Program of China (Grant No. 2015CB953904), the National Natural Science Foundation of China (Grant Nos. 11275072 and 11435005), the Doctoral Program of Higher Education of China (Grant No. 20120076110024), the Network Information Physics Calculation of Basic Research Innovation Research Group of China (Grant No. 61321064), and Shanghai Collaborative Innovation Center of Trustworthy Software for Internet of Things, China (Grant No. ZF1213).

Rate and Localization of Graft Detachment in Descemet Membrane Endothelial Keratoplasty.

PubMed

Maier, Anna-Karina B; Gundlach, Enken; Pilger, Daniel; Rübsam, Anne; Klamann, Matthias K J; Gonnermann, Johannes; Bertelmann, Eckart; Joussen, Antonia M; Torun, Necip

2016-03-01

To investigate the rate and localization of graft detachment after Descemet membrane endothelial keratoplasty. Sixty-six consecutive cases operated between June and August 2014 at the Charité-Universitätsmedizin Berlin were examined prospectively 1 week postoperatively. A single masked observer analyzed the rate and localization of graft detachment using optical coherence tomography (OCT), and the rebubbling rate was measured. Localization of graft detachment was correlated to the incision approach. Preoperative data were correlated to the rate of graft detachment and rebubbling. Graft detachment occurred in more than 2 clock hours and with postoperative corneal edema in 33.3% and required rebubbling. In 33.3%, graft detachment occurred in more than 2 clock hours and with postoperative corneal edema and required rebubbling. The mean graft detachment rate was 8.3% per clock hour. A significantly higher graft detachment rate was noted in the inferior clock hours (21.1%, P < 0.0001, 16.7%, P = 0.003). Only higher age of the patient correlated to a higher rate of graft detachment (P = 0.022). No correlation was found between localization of graft detachment and the incision approach (P = 0.615). The graft detachment rate is high after Descemet membrane endothelial keratoplasty, but detachment is usually peripheral, partial and mainly inferior and involves only a few clock hours. Only higher age of the patient is strongly associated with a higher rate of graft detachment. The incision approach is not significantly correlated with the localization of graft detachment. Therefore, the postoperative supine position of the patient seems to be of major importance.Clinical Trial Registration-URL: http://www.clinicaltrials.gov. Unique identifier: NCT02020044.

Membrane-localized ubiquitin ligase ATL15 functions in sugar-responsive growth regulation in Arabidopsis.

PubMed

Aoyama, Shoki; Terada, Saki; Sanagi, Miho; Hasegawa, Yoko; Lu, Yu; Morita, Yoshie; Chiba, Yukako; Sato, Takeo; Yamaguchi, Junji

2017-09-09

Ubiquitin ligases play important roles in regulating various cellular processes by modulating the protein function of specific ubiquitination targets. The Arabidopsis Tóxicos en Levadura (ATL) family is a group of plant-specific RING-type ubiquitin ligases that localize to membranes via their N-terminal transmembrane-like domains. To date, 91 ATL isoforms have been identified in the Arabidopsis genome, with several ATLs reported to be involved in regulating plant responses to environmental stresses. However, the functions of most ATLs remain unknown. This study, involving transcriptome database analysis, identifies ATL15 as a sugar responsive ATL gene in Arabidopsis. ATL15 expression was rapidly down-regulated in the presence of sugar. The ATL15 protein showed ubiquitin ligase activity in vitro and localized to plasma membrane and endomembrane compartments. Further genetic analyses demonstrated that the atl15 knockout mutants are insensitive to high glucose concentrations, whereas ATL15 overexpression depresses plant growth. In addition, endogenous glucose and starch amounts were reciprocally affected in the atl15 knockout mutants and the ATL15 overexpressors. These results suggest that ATL15 protein plays a significant role as a membrane-localized ubiquitin ligase that regulates sugar-responsive plant growth in Arabidopsis. Copyright © 2017 Elsevier Inc. All rights reserved.

A Novel Phosphatidylinositol 4,5-Bisphosphate Binding Domain Mediates Plasma Membrane Localization of ExoU and Other Patatin-like Phospholipases*

PubMed Central

Tyson, Gregory H.; Halavaty, Andrei S.; Kim, Hyunjin; Geissler, Brett; Agard, Mallory; Satchell, Karla J.; Cho, Wonhwa; Anderson, Wayne F.; Hauser, Alan R.

2015-01-01

Bacterial toxins require localization to specific intracellular compartments following injection into host cells. In this study, we examined the membrane targeting of a broad family of bacterial proteins, the patatin-like phospholipases. The best characterized member of this family is ExoU, an effector of the Pseudomonas aeruginosa type III secretion system. Upon injection into host cells, ExoU localizes to the plasma membrane, where it uses its phospholipase A2 activity to lyse infected cells. The targeting mechanism of ExoU is poorly characterized, but it was recently found to bind to the phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a marker for the plasma membrane of eukaryotic cells. We confirmed that the membrane localization domain (MLD) of ExoU had a direct affinity for PI(4,5)P2, and we determined that this binding was required for ExoU localization. Previously uncharacterized ExoU homologs from Pseudomonas fluorescens and Photorhabdus asymbiotica also localized to the plasma membrane and required PI(4,5)P2 for this localization. A conserved arginine within the MLD was critical for interaction of each protein with PI(4,5)P2 and for localization. Furthermore, we determined the crystal structure of the full-length P. fluorescens ExoU and found that it was similar to that of P. aeruginosa ExoU. Each MLD contains a four-helical bundle, with the conserved arginine exposed at its cap to allow for interaction with the negatively charged PI(4,5)P2. Overall, these findings provide a structural explanation for the targeting of patatin-like phospholipases to the plasma membrane and define the MLD of ExoU as a member of a new class of PI(4,5)P2 binding domains. PMID:25505182

Bi-directional exchange of membrane components occurs during co-culture of mesenchymal stem cells and nucleus pulposus cells.

PubMed

Strassburg, Sandra; Hodson, Nigel W; Hill, Patrick I; Richardson, Stephen M; Hoyland, Judith A

2012-01-01

Mesenchymal stem cell (MSC)-based therapies have been proposed as novel treatments for intervertebral disc (IVD) degeneration. We have previously demonstrated that when MSCs are co-cultured with nucleus pulposus (NP) cells with direct cell-cell contact, they differentiate along the NP lineage and simultaneously stimulate the degenerate NP cell population to regain a normal (non-degenerate) phenotype, an effect which requires cell-cell communication. However, the mechanisms by which NP cells and MSCs interact in this system are currently unclear. Thus, in this study we investigated a range of potential mechanisms for exchange of cellular components or information that may direct these changes, including cell fusion, gap-junctional communication and exchange of membrane components by direct transfer or via microvesicle formation. Flow cytometry of fluorescently labeled MSCs and NP cells revealed evidence of some cell fusion and formation of gapjunctions, although at the three timepoints studied these phenomena were detectable only in a small proportion of cells. While these mechanisms may play a role in cell-cell communication, the data suggests they are not the predominant mechanism of interaction. However, flow cytometry of fluorescently dual-labeled cells showed that extensive bi-directional transfer of membrane components is operational during direct co-culture of MSCs and NP cells. Furthermore, there was also evidence for secretion and internalization of membrane-bound microvesicles by both cell types. Thus, this study highlights bi-directional intercellular transfer of membrane components as a possible mechanism of cellular communication between MSC and NP cells.

Influences of acid-base property of membrane on interfacial interactions related with membrane fouling in a membrane bioreactor based on thermodynamic assessment.

PubMed

Zhao, Leihong; Qu, Xiaolu; Zhang, Meijia; Lin, Hongjun; Zhou, Xiaoling; Liao, Bao-Qiang; Mei, Rongwu; Hong, Huachang

2016-08-01

Failure of membrane hydrophobicity in predicting membrane fouling requires a more reliable indicator. In this study, influences of membrane acid base (AB) property on interfacial interactions in two different interaction scenarios in a submerged membrane bioreactor (MBR) were studied according to thermodynamic approaches. It was found that both the polyvinylidene fluoride (PVDF) membrane and foulant samples in the MBR had relatively high electron donor (γ(-)) component and low electron acceptor (γ(+)) component. For both of interaction scenarios, AB interaction was the major component of the total interaction. The results showed that, the total interaction monotonically decreased with membrane γ(-), while was marginally affected by membrane γ(+), suggesting that γ(-) could act as a reliable indicator for membrane fouling prediction. This study suggested that membrane modification for fouling mitigation should orient to improving membrane surface γ(-) component rather than hydrophilicity. Copyright © 2016 Elsevier Ltd. All rights reserved.

Adaptive Decomposition of Highly Resolved Time Series into Local and Non‐local Components

EPA Science Inventory

Highly time-resolved air monitoring data are widely being collected over long time horizons in order to characterizeambient and near-source air quality trends. In many applications, it is desirable to split the time-resolved data into two ormore components (e.g., local and region...

Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane.

PubMed

Boassa, Daniela; Nguyen, Phuong; Hu, Junru; Ellisman, Mark H; Sosinsky, Gina E

2014-01-01

Pannexin2 (Panx2) is the largest of three members of the pannexin proteins. Pannexins are topologically related to connexins and innexins, but serve different functional roles than forming gap junctions. We previously showed that pannexins form oligomeric channels but unlike connexins and innexins, they form only single membrane channels. High levels of Panx2 mRNA and protein in the Central Nervous System (CNS) have been documented. Whereas Pannexin1 (Panx1) is fairly ubiquitous and Pannexin3 (Panx3) is found in skin and connective tissue, both are fully glycosylated, traffic to the plasma membrane and have functions correlated with extracellular ATP release. Here, we describe trafficking and subcellular localizations of exogenous Panx2 and Panx1 protein expression in MDCK, HeLa, and HEK 293T cells as well as endogenous Panx1 and Panx2 patterns in the CNS. Panx2 was found in intracellular localizations, was partially N-glycosylated, and localizations were non-overlapping with Panx1. Confocal images of hippocampal sections immunolabeled for the astrocytic protein GFAP, Panx1 and Panx2 demonstrated that the two isoforms, Panx1 and Panx2, localized at different subcellular compartments in both astrocytes and neurons. Using recombinant fusions of Panx2 with appended genetic tags developed for correlated light and electron microscopy and then expressed in different cell lines, we determined that Panx2 is localized in the membrane of intracellular vesicles and not in the endoplasmic reticulum as initially indicated by calnexin colocalization experiments. Dual immunofluorescence imaging with protein markers for specific vesicle compartments showed that Panx2 vesicles are early endosomal in origin. In electron tomographic volumes, cross-sections of these vesicles displayed fine structural details and close proximity to actin filaments. Thus, pannexins expressed at different subcellular compartments likely exert distinct functional roles, particularly in the nervous system.

Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

PubMed Central

Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

1996-01-01

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

Biochemical characterization of detergent-resistant membranes: a systematic approach

PubMed Central

Babiychuk, Eduard B.; Draeger, Annette

2006-01-01

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be ‘investigator-specific’. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the ‘brick-like’ raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data. PMID:16608442

Biochemical characterization of detergent-resistant membranes: a systematic approach.

PubMed

Babiychuk, Eduard B; Draeger, Annette

2006-08-01

Lateral segregation of cholesterol- and sphingomyelin-rich rafts and glycerophospholipid-containing non-raft microdomains has been proposed to play a role in a variety of biological processes. The most compelling evidence for membrane segregation is based on the observation that extraction with non-ionic detergents leads to solubilization of a subset of membrane components only. However, one decade later, a large body of inconsistent detergent-extraction data is threatening the very concept of membrane segregation. We have assessed the validity of the existing paradigms and we show the following. (i) The localization of a membrane component within a particular fraction of a sucrose gradient cannot be taken as a yardstick for its solubility: a variable localization of the DRMs (detergent-resistant membranes) in sucrose gradients is the result of complex associations between the membrane skeleton and the lipid bilayer. (ii) DRMs of variable composition can be generated by using a single detergent, the increasing concentration of which gradually extracts one protein/lipid after another. Therefore any extraction pattern obtained by a single concentration experiment is bound to be 'investigator-specific'. It follows that comparison of DRMs obtained by different detergents in a single concentration experiment is prone to misinterpretations. (iii) Depletion of cholesterol has a graded effect on membrane solubility. (iv) Differences in detergent solubility of the members of the annexin protein family arise from their association with chemically different membrane compartments; however, these cannot be attributed to the 'brick-like' raft-building blocks of fixed size and chemical composition. Our findings demonstrate a need for critical re-evaluation of the accumulated detergent-extraction data.

Interaction of miltefosine with the lipid and protein components of the erythrocyte membrane.

PubMed

Moreira, Rodrigo Alves; Mendanha, Sebastião Antonio; Hansen, Daiane; Alonso, Antonio

2013-05-01

Miltefosine (MT) is an alkylphospholipid that has been approved for the treatment of breast cancer metastasis and visceral leishmaniasis, although its mechanism of action remains poorly understood. Electron paramagnetic resonance spectroscopy of a spin-labeled lipid and a thiol-specific spin label showed that MT causes an increase in the molecular dynamics of erythrocyte ghost membranes and detergent-resistant membranes (DRMs) prepared from erythrocyte ghosts. In the vesicles of lipid raft constituents, it was shown that 20 mol % sphingomyelin could be replaced by 20 mol % MT with no change in the molecular dynamics. The effect of MT in DRMs was more pronounced than in erythrocyte ghosts, supporting the hypothesis that MT is a lipid raft modulator. At the reported MT-plasma concentrations found during the treatment of leishmaniasis (31-90 µg/mL), our measurements in the blood plasma indicated a hemolytic level of 2%-5%. The experiments indicated that MT acts predominantly on the protein component of the membrane. MT aggregates may wrap around the hydrophobic polypeptide chains, forming micelle-like structures that stabilize protein conformations more exposed to the solvent. Proteins with higher hydrophobicity may induce the penetration of the hydrophilic groups of MT into the membrane and cause it to rupture. Copyright © 2013 Wiley Periodicals, Inc.

Annexin A2 Mediates the Localization of Measles Virus Matrix Protein at the Plasma Membrane.

PubMed

Koga, Ritsuko; Kubota, Marie; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-02-28

Annexins are a family of structurally related proteins that bind negatively charged membrane phospholipids in a Ca 2+ -dependent manner. Annexin A2 (AnxA2), a member of the family, has been implicated in a variety of cellular functions including the organization of membrane domains, vesicular trafficking and cell-cell adhesion. AnxA2 generally forms the heterotetrameric complex with a small Ca 2+ -binding protein S100A10. Measles virus (MV), a member of the family Paramyxoviridae , is an enveloped virus with a nonsegmented negative strand RNA genome. Knockdown of AnxA2 greatly reduced MV growth in cells, without affecting its entry and viral RNA production. In MV-infected, AnxA2-knockdown cells, the expression level of the matrix (M) protein, but not other viral proteins, was reduced compared with that in control cells, and the distribution of the M protein at the plasma membrane was decreased. The M protein lines the inner surface of the envelope and plays an important role in virus assembly by connecting the nucleocapsid to the envelope proteins. The M protein bound to AnxA2 independently of AnxA2's phosphorylation or its association with S100A10, and was co-localized with AnxA2 within cells. Truncation of the N-terminal 10 amino acid residues, but not the N-terminal 5 residues, compromised the ability of the M protein to interact with AnxA2 and localize at the plasma membrane. These results indicate that AnxA2 mediates the localization of the MV M protein at the plasma membrane by interacting with its N-terminal region (especially residues at positions 6-10), thereby aiding in MV assembly. IMPORTANCE Measles virus (MV) is an important human pathogen, still claiming ∼ 100,000 lives per year despite the presence of effective vaccines, and causes occasional outbreaks even in developed countries. Replication of viruses largely relies on the functions of host cells. Our study revealed that the reduction of the host protein annexin A2 compromises the replication of

S-Acylation of the cellulose synthase complex is essential for its plasma membrane localization.

PubMed

Kumar, Manoj; Wightman, Raymond; Atanassov, Ivan; Gupta, Anjali; Hurst, Charlotte H; Hemsley, Piers A; Turner, Simon

2016-07-08

Plant cellulose microfibrils are synthesized by a process that propels the cellulose synthase complex (CSC) through the plane of the plasma membrane. How interactions between membranes and the CSC are regulated is currently unknown. Here, we demonstrate that all catalytic subunits of the CSC, known as cellulose synthase A (CESA) proteins, are S-acylated. Analysis of Arabidopsis CESA7 reveals four cysteines in variable region 2 (VR2) and two cysteines at the carboxy terminus (CT) as S-acylation sites. Mutating both the VR2 and CT cysteines permits CSC assembly and trafficking to the Golgi but prevents localization to the plasma membrane. Estimates suggest that a single CSC contains more than 100 S-acyl groups, which greatly increase the hydrophobic nature of the CSC and likely influence its immediate membrane environment. Copyright © 2016, American Association for the Advancement of Science.

MHC Class II and CD9 in human eosinophils localize to detergent-resistant membrane microdomains.

PubMed

Akuthota, Praveen; Melo, Rossana C N; Spencer, Lisa A; Weller, Peter F

2012-02-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor-stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR-containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4(+) T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils.

MHC Class II and CD9 in Human Eosinophils Localize to Detergent-Resistant Membrane Microdomains

PubMed Central

Akuthota, Praveen; Melo, Rossana C. N.; Spencer, Lisa A.

2012-01-01

Eosinophils function in murine allergic airways inflammation as professional antigen-presenting cells (APCs). In murine professional APC cell types, optimal functioning of MHC Class II depends on its lateral association in plasma membranes and colocalization with the tetraspanin CD9 into detergent-resistant membrane microdomains (DRMs). With human eosinophils, we evaluated the localization of MHC Class II (HLA-DR) to DRMs and the functional significance of such localization. In granulocyte-macrophage colony-stimulating factor–stimulated human eosinophils, antibody cross-linked HLA-DR colocalized by immunofluorescence microscopy focally on plasma membranes with CD9 and the DRM marker ganglioside GM1. In addition, HLA-DR coimmunoprecipitates with CD9 after chemical cross-linking of CD9. HLA-DR and CD9 were localized by Western blotting in eosinophil DRM subcellular fractions. DRM disruption with the cholesterol-depleting agent methyl-β-cyclodextrin decreased eosinophil surface expression of HLA-DR and CD9. We show that CD9 is abundant on the surface of eosinophils, presenting the first electron microscopy data of the ultrastructural immunolocalization of CD9 in human eosinophils. Disruption of HLA-DR–containing DRMs decreased the ability of superantigen-loaded human eosinophils to stimulate CD4+ T-cell activation (CD69 expression), proliferation, and cytokine production. Our results, which demonstrate that eosinophil MHC Class II localizes to DRMs in association with CD9 in a functionally significant manner, represent a novel insight into the organization of the antigen presentation complex of human eosinophils. PMID:21885678

Overview of online two-dimensional liquid chromatography based on cell membrane chromatography for screening target components from traditional Chinese medicines.

PubMed

Muhammad, Saqib; Han, Shengli; Xie, Xiaoyu; Wang, Sicen; Aziz, Muhammad Majid

2017-01-01

Cell membrane chromatography is a simple, specific, and time-saving technique for studying drug-receptor interactions, screening of active components from complex mixtures, and quality control of traditional Chinese medicines. However, the short column life, low sensitivity, low column efficiency (so cannot resolve satisfactorily mixture of compounds), low peak capacity, and inefficient in structure identification were bottleneck in its application. Combinations of cell membrane chromatography with multidimensional chromatography such as two-dimensional liquid chromatography and high sensitivity detectors like mass have significantly reduced many of the above-mentioned shortcomings. This paper provides an overview of the current advances in online two-dimensional-based cell membrane chromatography for screening target components from traditional Chinese medicines with particular emphasis on the instrumentation, preparation of cell membrane stationary phase, advantages, and disadvantages compared to alternative approaches. The last section of the review summarizes the applications of the online two-dimensional high-performance liquid chromatography based cell membrane chromatography reported since its emergence to date (2010-June 2016). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

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

Clarke, J.T.; Cook, H.W.; Spence, M.W.

1985-03-01

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22 h in the presence of D-(1-/sup 3/H)galactose or (/sup 3/H)GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipid-sialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellularmore » membrane fractions studied was recovered from plasma membrane and only 10-15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous (/sup 3/H)GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.« less

Prostate Specific Membrane Antigen Positron Emission Tomography May Improve the Diagnostic Accuracy of Multiparametric Magnetic Resonance Imaging in Localized Prostate Cancer.

PubMed

Rhee, H; Thomas, P; Shepherd, B; Gustafson, S; Vela, I; Russell, P J; Nelson, C; Chung, E; Wood, G; Malone, G; Wood, S; Heathcote, P

2016-10-01

Positron emission tomography using ligands targeting prostate specific membrane antigen has recently been introduced. Positron emission tomography imaging with (68)Ga-PSMA-HBED-CC has been shown to detect metastatic prostate cancer lesions at a high rate. In this study we compare multiparametric magnetic resonance imaging and prostate specific membrane antigen positron emission tomography of the prostate with whole mount ex vivo prostate histopathology to determine the true sensitivity and specificity of these imaging modalities for detecting and locating tumor foci within the prostate. In a prospective clinical trial setting 20 patients with localized prostate cancer and a planned radical prostatectomy were recruited. All patients underwent multiparametric magnetic resonance imaging and positron emission tomography before surgery, and whole mount histopathology slides were directly compared to the images. European Society of Urogenital Radiology guidelines for reporting magnetic resonance imaging were used as a template for regional units of analysis. The uropathologist and radiologists were blinded to individual components of the study, and the final correlation was performed by visual and deformable registration analysis. A total of 50 clinically significant lesions were identified from the whole mount histopathological analysis. Based on regional analysis the sensitivity, specificity, positive predictive value and negative predictive value for multiparametric magnetic resonance imaging were 44%, 94%, 81% and 76%, respectively. With prostate specific membrane antigen positron emission tomography the sensitivity, specificity, positive predictive value and negative predictive value were 49%, 95%, 85% and 88%, respectively. Prostate specific membrane antigen positron emission tomography yielded a higher specificity and positive predictive value. A significant proportion of cancers are potentially missed and underestimated by both imaging modalities. Prostate

Atomistic Monte Carlo Simulation of Lipid Membranes

PubMed Central

Wüstner, Daniel; Sklenar, Heinz

2014-01-01

Biological membranes are complex assemblies of many different molecules of which analysis demands a variety of experimental and computational approaches. In this article, we explain challenges and advantages of atomistic Monte Carlo (MC) simulation of lipid membranes. We provide an introduction into the various move sets that are implemented in current MC methods for efficient conformational sampling of lipids and other molecules. In the second part, we demonstrate for a concrete example, how an atomistic local-move set can be implemented for MC simulations of phospholipid monomers and bilayer patches. We use our recently devised chain breakage/closure (CBC) local move set in the bond-/torsion angle space with the constant-bond-length approximation (CBLA) for the phospholipid dipalmitoylphosphatidylcholine (DPPC). We demonstrate rapid conformational equilibration for a single DPPC molecule, as assessed by calculation of molecular energies and entropies. We also show transition from a crystalline-like to a fluid DPPC bilayer by the CBC local-move MC method, as indicated by the electron density profile, head group orientation, area per lipid, and whole-lipid displacements. We discuss the potential of local-move MC methods in combination with molecular dynamics simulations, for example, for studying multi-component lipid membranes containing cholesterol. PMID:24469314

A modular platform for one-step assembly of multi-component membrane systems by fusion of charged proteoliposomes

NASA Astrophysics Data System (ADS)

Ishmukhametov, Robert R.; Russell, Aidan N.; Berry, Richard M.

2016-10-01

An important goal in synthetic biology is the assembly of biomimetic cell-like structures, which combine multiple biological components in synthetic lipid vesicles. A key limiting assembly step is the incorporation of membrane proteins into the lipid bilayer of the vesicles. Here we present a simple method for delivery of membrane proteins into a lipid bilayer within 5 min. Fusogenic proteoliposomes, containing charged lipids and membrane proteins, fuse with oppositely charged bilayers, with no requirement for detergent or fusion-promoting proteins, and deliver large, fragile membrane protein complexes into the target bilayers. We demonstrate the feasibility of our method by assembling a minimal electron transport chain capable of adenosine triphosphate (ATP) synthesis, combining Escherichia coli F1Fo ATP-synthase and the primary proton pump bo3-oxidase, into synthetic lipid vesicles with sizes ranging from 100 nm to ~10 μm. This provides a platform for the combination of multiple sets of membrane protein complexes into cell-like artificial structures.

Identification of the components of a glycolytic enzyme metabolon on the human red blood cell membrane.

PubMed

Puchulu-Campanella, Estela; Chu, Haiyan; Anstee, David J; Galan, Jacob A; Tao, W Andy; Low, Philip S

2013-01-11

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as "label transfer" that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction.

Identification of the Components of a Glycolytic Enzyme Metabolon on the Human Red Blood Cell Membrane*

PubMed Central

Puchulu-Campanella, Estela; Chu, Haiyan; Anstee, David J.; Galan, Jacob A.; Tao, W. Andy; Low, Philip S.

2013-01-01

Glycolytic enzymes (GEs) have been shown to exist in multienzyme complexes on the inner surface of the human erythrocyte membrane. Because no protein other than band 3 has been found to interact with GEs, and because several GEs do not bind band 3, we decided to identify the additional membrane proteins that serve as docking sites for GE on the membrane. For this purpose, a method known as “label transfer” that employs a photoactivatable trifunctional cross-linking reagent to deliver a biotin from a derivatized GE to its binding partner on the membrane was used. Mass spectrometry analysis of membrane proteins that were biotinylated following rebinding and photoactivation of labeled GAPDH, aldolase, lactate dehydrogenase, and pyruvate kinase revealed not only the anticipated binding partner, band 3, but also the association of GEs with specific peptides in α- and β-spectrin, ankyrin, actin, p55, and protein 4.2. More importantly, the labeled GEs were also found to transfer biotin to other GEs in the complex, demonstrating for the first time that GEs also associate with each other in their membrane complexes. Surprisingly, a new GE binding site was repeatedly identified near the junction of the membrane-spanning and cytoplasmic domains of band 3, and this binding site was confirmed by direct binding studies. These results not only identify new components of the membrane-associated GE complexes but also provide molecular details on the specific peptides that form the interfacial contacts within each interaction. PMID:23150667

Sentan: A Novel Specific Component of the Apical Structure of Vertebrate Motile Cilia

PubMed Central

Yuba-Kubo, Akiko; Tsukita, Sachiko; Tsukita, Shoichiro; Amagai, Masayuki

2008-01-01

Human respiratory and oviductal cilia have specific apical structures characterized by a narrowed distal portion and a ciliary crown. These structures are conserved among vertebrates that have air respiration systems; however, the molecular components of these structures have not been defined, and their functions are unknown. To identify the molecular component(s) of the cilia apical structure, we screened EST libraries to identify gene(s) that are exclusively expressed in ciliated tissues, are transcriptionally up-regulated during in vitro ciliogenesis, and are not expressed in testis (because sperm flagella have no such apical structures). One of the identified gene products, named sentan, was localized to the distal tip region of motile cilia. Using anti-sentan polyclonal antibodies and electron microscopy, sentan was shown to localize exclusively to the bridging structure between the cell membrane and peripheral singlet microtubules, which specifically exists in the narrowed distal portion of cilia. Exogenously expressed sentan showed affinity for the membrane protrusions, and a protein–lipid binding assay revealed that sentan bound to phosphatidylserine. These findings suggest that sentan is the first molecular component of the ciliary tip to bridge the cell membrane and peripheral singlet microtubules, making the distal portion of the cilia narrow and stiff to allow for better airway clearance or ovum transport. PMID:18829862

Transmembrane protein PERP is a component of tessellate junctions and of other junctional and non-junctional plasma membrane regions in diverse epithelial and epithelium-derived cells.

PubMed

Franke, Werner W; Heid, Hans; Zimbelmann, Ralf; Kuhn, Caecilia; Winter-Simanowski, Stefanie; Dörflinger, Yvette; Grund, Christine; Rickelt, Steffen

2013-07-01

Protein PERP (p53 apoptosis effector related to PMP-22) is a small (21.4 kDa) transmembrane polypeptide with an amino acid sequence indicative of a tetraspanin character. It is enriched in the plasma membrane and apparently contributes to cell-cell contacts. Hitherto, it has been reported to be exclusively a component of desmosomes of some stratified epithelia. However, by using a series of newly generated mono- and polyclonal antibodies, we show that protein PERP is not only present in all kinds of stratified epithelia but also occurs in simple, columnar, complex and transitional epithelia, in various types of squamous metaplasia and epithelium-derived tumors, in diverse epithelium-derived cell cultures and in myocardial tissue. Immunofluorescence and immunoelectron microscopy allow us to localize PERP predominantly in small intradesmosomal locations and in variously sized, junction-like peri- and interdesmosomal regions ("tessellate junctions"), mostly in mosaic or amalgamated combinations with other molecules believed, to date, to be exclusive components of tight and adherens junctions. In the heart, PERP is a major component of the composite junctions of the intercalated disks connecting cardiomyocytes. Finally, protein PERP is a cobblestone-like general component of special plasma membrane regions such as the bile canaliculi of liver and subapical-to-lateral zones of diverse columnar epithelia and upper urothelial cell layers. We discuss possible organizational and architectonic functions of protein PERP and its potential value as an immunohistochemical diagnostic marker.

Characterization of membrane association of Rinderpest virus matrix protein

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

Subhashri, R.; Shaila, M.S.

2007-04-20

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M proteinmore » gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein.« less

Controlling Cargo Trafficking in Multicomponent Membranes.

PubMed

Curk, Tine; Wirnsberger, Peter; Dobnikar, Jure; Frenkel, Daan; Šarić, Anđela

2018-04-27

Biological membranes typically contain a large number of different components dispersed in small concentrations in the main membrane phase, including proteins, sugars, and lipids of varying geometrical properties. Most of these components do not bind the cargo. Here, we show that such "inert" components can be crucial for the precise control of cross-membrane trafficking. Using a statistical mechanics model and molecular dynamics simulations, we demonstrate that the presence of inert membrane components of small isotropic curvatures dramatically influences cargo endocytosis, even if the total spontaneous curvature of such a membrane remains unchanged. Curved lipids, such as cholesterol, as well as asymmetrically included proteins and tethered sugars can, therefore, actively participate in the control of the membrane trafficking of nanoscopic cargo. We find that even a low-level expression of curved inert membrane components can determine the membrane selectivity toward the cargo size and can be used to selectively target membranes of certain compositions. Our results suggest a robust and general method of controlling cargo trafficking by adjusting the membrane composition without needing to alter the concentration of receptors or the average membrane curvature. This study indicates that cells can prepare for any trafficking event by incorporating curved inert components in either of the membrane leaflets.

Molecular counting of membrane receptor subunits with single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Krüger, Carmen; Fricke, Franziska; Karathanasis, Christos; Dietz, Marina S.; Malkusch, Sebastian; Hummer, Gerhard; Heilemann, Mike

2017-02-01

We report on quantitative single-molecule localization microscopy, a method that next to super-resolved images of cellular structures provides information on protein copy numbers in protein clusters. This approach is based on the analysis of blinking cycles of single fluorophores, and on a model-free description of the distribution of the number of blinking events. We describe the experimental and analytical procedures, present cellular data of plasma membrane proteins and discuss the applicability of this method.

Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization

PubMed Central

Turnbull, Andrew P; Kümmel, Daniel; Prinz, Bianka; Holz, Caterina; Schultchen, Jeffrey; Lang, Christine; Niesen, Frank H; Hofmann, Klaus-Peter; Delbrück, Heinrich; Behlke, Joachim; Müller, Eva-Christina; Jarosch, Ernst; Sommer, Thomas; Heinemann, Udo

2005-01-01

BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-Å resolution. BET3 adopts an α/β-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein–protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions. PMID:15692564

Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane

PubMed Central

Lei, Jennifer; Priddy, Lauren B.; Lim, Jeremy J.; Massee, Michelle; Koob, Thomas J.

2017-01-01

Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION® Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek® activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair. PMID:28224047

Identification of Extracellular Matrix Components and Biological Factors in Micronized Dehydrated Human Amnion/Chorion Membrane.

PubMed

Lei, Jennifer; Priddy, Lauren B; Lim, Jeremy J; Massee, Michelle; Koob, Thomas J

2017-02-01

Objective: The use of bioactive extracellular matrix (ECM) grafts such as amniotic membranes is an attractive treatment option for enhancing wound repair. In this study, the concentrations, activity, and distribution of matrix components, growth factors, proteases, and inhibitors were evaluated in PURION ® Processed, micronized, dehydrated human amnion/chorion membrane (dHACM; MiMedx Group, Inc.). Approach: ECM components in dHACM tissue were assessed by using immunohistochemical staining, and growth factors, cytokines, proteases, and inhibitors were quantified by using single and multiplex ELISAs. The activities of proteases that were native to the tissue were determined via gelatin zymography and EnzChek ® activity assay. Results: dHACM tissue contained the ECM components collagens I and IV, hyaluronic acid, heparin sulfate proteoglycans, fibronectin, and laminin. In addition, numerous growth factors, cytokines, chemokines, proteases, and protease inhibitors that are known to play a role in the wound-healing process were quantified in dHACM. Though matrix metalloproteinases (MMPs) were present in dHACM tissues, inhibitors of MMPs overwhelmingly outnumbered the MMP enzymes by an overall molar ratio of 28:1. Protease activity assays revealed that the MMPs in the tissue existed primarily either in their latent form or complexed with inhibitors. Innovation: This is the first study to characterize components that function in wound healing, including inhibitor and protease content and activity, in micronized dHACM. Conclusion: A variety of matrix components and growth factors, as well as proteases and their inhibitors, were identified in micronized dHACM, providing a better understanding of how micronized dHACM tissue can be used to effectively promote wound repair.

Localization and proteomic characterization of cholesterol-rich membrane microdomains in the inner ear.

PubMed

Thomas, Paul V; Cheng, Andrew L; Colby, Candice C; Liu, Liqian; Patel, Chintan K; Josephs, Lydia; Duncan, R Keith

2014-05-30

Biological membranes organize and compartmentalize cell signaling into discrete microdomains, a process that often involves stable, cholesterol-rich platforms that facilitate protein-protein interactions. Polarized cells with distinct apical and basolateral cell processes rely on such compartmentalization to maintain proper function. In the cochlea, a variety of highly polarized sensory and non-sensory cells are responsible for the early stages of sound processing in the ear, yet little is known about the mechanisms that traffic and organize signaling complexes within these cells. We sought to determine the prevalence, localization, and protein composition of cholesterol-rich lipid microdomains in the cochlea. Lipid raft components, including the scaffolding protein caveolin and the ganglioside GM1, were found in sensory, neural, and glial cells. Mass spectrometry of detergent-resistant membrane (DRM) fractions revealed over 600 putative raft proteins associated with subcellular localization, trafficking, and metabolism. Among the DRM constituents were several proteins involved in human forms of deafness including those involved in ion homeostasis, such as the potassium channel KCNQ1, the co-transporter SLC12A2, and gap junction proteins GJA1 and GJB6. The presence of caveolin in the cochlea and the abundance of proteins in cholesterol-rich DRM suggest that lipid microdomains play a significant role in cochlear physiology. Although mechanisms underlying cholesterol synthesis, homeostasis, and compartmentalization in the ear are poorly understood, there are several lines of evidence indicating that cholesterol is a key modulator of cochlear function. Depletion of cholesterol in mature sensory cells alters calcium signaling, changes excitability during development, and affects the biomechanical processes in outer hair cells that are responsible for hearing acuity. More recently, we have established that the cholesterol-modulator beta-cyclodextrin is capable of

Organic fluid permeation through fluoropolymer membranes

DOEpatents

Nemser, Stuart M.; Kosaraju, Praveen; Bowser, John

2015-07-14

Separation of the components of liquid mixtures is achieved by contacting a liquid mixture with a nonporous membrane having a fluoropolymer selectively permeable layer and imposing a pressure gradient across the membrane from feed side to permeate side. Unusually high transmembrane flux is obtained when the membrane is subjected to one or more process conditions prior to separation. These include (a) leaving some residual amount of membrane casting solvent in the membrane, and (b) contacting the membrane with a component of the mixture to be separated for a duration effective to saturate the membrane with the component.

The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition.

PubMed

Ishida, Hiroaki; Garcia-Herrero, Alicia; Vogel, Hans J

2014-12-01

Gram-negative bacteria such as Escherichia coli are surrounded by two membranes with a thin peptidoglycan (PG)-layer located in between them in the periplasmic space. The outer membrane protein A (OmpA) is a 325-residue protein and it is the major protein component of the outer membrane of E. coli. Previous structure determinations have focused on the N-terminal fragment (residues 1-171) of OmpA, which forms an eight stranded transmembrane β-barrel in the outer membrane. Consequently it was suggested that OmpA is composed of two independently folded domains in which the N-terminal β-barrel traverses the outer membrane and the C-terminal domain (residues 180-325) adopts a folded structure in the periplasmic space. However, some reports have proposed that full-length OmpA can instead refold in a temperature dependent manner into a single domain forming a larger transmembrane pore. Here, we have determined the NMR solution structure of the C-terminal periplasmic domain of E. coli OmpA (OmpA(180-325)). Our structure reveals that the C-terminal domain folds independently into a stable globular structure that is homologous to the previously reported PG-associated domain of Neisseria meningitides RmpM. Our results lend credence to the two domain structure model and a PG-binding function for OmpA, and we could indeed localize the PG-binding site on the protein through NMR chemical shift perturbation experiments. On the other hand, we found no evidence for binding of OmpA(180-325) with the TonB protein. In addition, we have also expressed and purified full-length OmpA (OmpA(1-325)) to study the structure of the full-length protein in micelles and nanodiscs by NMR spectroscopy. In both membrane mimetic environments, the recombinant OmpA maintains its two domain structure that is connected through a flexible linker. A series of temperature-dependent HSQC experiments and relaxation dispersion NMR experiments detected structural destabilization in the bulge region of the

Local-Level Prognostics Health Management Systems Framework for Passive AdvSMR Components. Interim Report

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

Ramuhalli, Pradeep; Roy, Surajit; Hirt, Evelyn H.

2014-09-12

This report describes research results to date in support of the integration and demonstration of diagnostics technologies for prototypical AdvSMR passive components (to establish condition indices for monitoring) with model-based prognostics methods. The focus of the PHM methodology and algorithm development in this study is at the localized scale. Multiple localized measurements of material condition (using advanced nondestructive measurement methods), along with available measurements of the stressor environment, enhance the performance of localized diagnostics and prognostics of passive AdvSMR components and systems.

Effect of a Vietnamese Cinnamomum cassia essential oil and its major component trans-cinnamaldehyde on the cell viability, membrane integrity, membrane fluidity, and proton motive force of Listeria innocua.

PubMed

Trinh, Nga-Thi-Thanh; Dumas, Emilie; Thanh, Mai Le; Degraeve, Pascal; Ben Amara, Chedia; Gharsallaoui, Adem; Oulahal, Nadia

2015-04-01

The antibacterial mechanism of a Cinnamomum cassia essential oil from Vietnam and of its main component (trans-cinnamaldehyde, 90% (m/m) of C. cassia essential oil) against a Listeria innocua strain was investigated to estimate their potential for food preservation. In the presence of C. cassia essential oil or trans-cinnamaldehyde at their minimal bactericidal concentration (2700 μg·mL(-1)), L. innocua cells fluoresced green after staining with Syto9® and propidium iodide, as observed by epifluorescence microscopy, suggesting that the perturbation of membrane did not cause large pore formation and cell lysis but may have introduced the presence of viable but nonculturable bacteria. Moreover, the fluidity, potential, and intracellular pH of the cytoplasmic membrane were perturbed in the presence of the essential oil or trans-cinnamaldehyde. However, these membrane perturbations were less severe in the presence of trans-cinnamaldehyde than in the presence of multicomponent C. cassia essential oil. This indicates that in addition to trans-cinnamaldehyde, other minor C. cassia essential oil components play a major role in its antibacterial activity against L. innocua cells.

Solid-state membrane module

DOEpatents

Gordon, John Howard [Salt Lake City, UT; Taylor, Dale M [Murray, UT

2011-06-07

Solid-state membrane modules comprising at least one membrane unit, where the membrane unit has a dense mixed conducting oxide layer, and at least one conduit or manifold wherein the conduit or manifold comprises a dense layer and at least one of a porous layer and a slotted layer contiguous with the dense layer. The solid-state membrane modules may be used to carry out a variety of processes including the separating of any ionizable component from a feedstream wherein such ionizable component is capable of being transported through a dense mixed conducting oxide layer of the membrane units making up the membrane modules. For ease of construction, the membrane units may be planar.

Damage of Escherichia coli membrane by bactericidal agent polyhexamethylene guanidine hydrochloride: micrographic evidences.

PubMed

Zhou, Z X; Wei, D F; Guan, Y; Zheng, A N; Zhong, J J

2010-03-01

The purpose of this study was to provide micrographic evidences for the damaged membrane structure and intracellular structure change of Escherichia coli strain 8099, induced by polyhexamethylene guanidine hydrochloride (PHMG). The bactericidal effect of PHMG on E. coli was investigated based on beta-galactosidase activity assay, fluorescein-5-isothiocyanate confocal laser scanning microscopy, field emission scanning electron microscopy and transmission electron microscopy. The results revealed that a low dose (13 microg ml(-1)) of PHMG slightly damaged the outer membrane structure of the treated bacteria and increased the permeability of the cytoplasmic membrane, while no significant damage was observed to the morphological structure of the cells. A high dose (23 microg ml(-1)) of PHMG collapsed the outer membrane structure, led to the formation of a local membrane pore across the membrane and badly damaged the internal structure of the cells. Subsequently, intracellular components were leaked followed by cell inactivation. Dose-dependent membrane disruption was the main bactericidal mechanism of PHMG. The formation of the local membrane pores was probable after exposure to a high dose (23 microg ml(-1)) of PHMG. Micrographic evidences were provided about the damaged membrane structure and intracellular structure change of E. coli. The presented information helps understand the bactericidal mechanism of PHMG by membrane damage.

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

PubMed Central

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

2016-01-01

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

Six components observations of local earthquakes during the 2016 Central Italy seismic sequence

NASA Astrophysics Data System (ADS)

Simonelli, A.; Bernauer, F.; Chow, B.; Braun, T.; Wassermann, J. M.; Igel, H.

2017-12-01

For many years the seismological community has looked for a reliable, sensitive, broadband three-component portable rotational sensor. In this preliminary study, we show the possibility of measuring and extracting relevant seismological information from local earthquakes. We employ portable three-component rotational sensors, insensitive to translations, which operate on optical interferometry principles (Sagnac effect). Multiple sensors recording redundantly add significance to the measurements.During the Central Italy seismic sequence in November 2016, we deployed two portable fiber-optic gyroscopes (BlueSeis3A from iXBlue and LCG demonstrator from LITEF) and a broadband seismometer in Colfiorito, Italy. We present here the six-component observations, with analysis of rotational (three redundant components) and translational (three components) ground motions, generated by earthquakes at local distances. For each seismic event, we compare coherence between rotational sensors and estimate a back azimuth consistent with theoretical values. We also estimate Love and Rayleigh wave phase velocities in the 5 to 10 Hz frequency range.

The Pro-Apoptotic BH3-Only Protein Bim Interacts with Components of the Translocase of the Outer Mitochondrial Membrane (TOM)

PubMed Central

Frank, Daniel O.; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

2015-01-01

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated. PMID:25875815

The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

PubMed

Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

2015-01-01

The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

Large effect of membrane tension on the fluid-solid phase transitions of two-component phosphatidylcholine vesicles.

PubMed

Chen, Dong; Santore, Maria M

2014-01-07

Model phospholipid membranes and vesicles have long provided insight into the nature of confined materials and membranes while also providing a platform for drug delivery. The rich thermodynamic behavior and interesting domain shapes in these membranes have previously been mapped in extensive studies that vary temperature and composition; however, the thermodynamic impact of tension on bilayers has been restricted to recent reports of subtly reduced fluid-fluid transition temperatures. In two-component phosphatidylcholine unilamellar vesicles [1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)], we report a dramatic influence of tension on the fluid-solid transition and resulting phases: At fixed composition, systematic variations in tension produce differently shaped solid domains (striped or irregular hexagons), shift fluid-solid transition temperatures, and produce a triple-point-like intersection of coexistence curves at elevated tensions, about 3 mN/m for 30% DOPC/70% DPPC. Tension therefore represents a potential switch of microstructure in responsive engineered materials; it is an important morphology-determining variable in confined systems, and, in biological membranes, it may provide a means to regulate dynamic structure.

Membrane Curvature Sensing by Amphipathic Helices

PubMed Central

Jensen, Martin Borch; Bhatia, Vikram Kjøller; Jao, Christine C.; Rasmussen, Jakob Ewald; Pedersen, Søren L.; Jensen, Knud J.; Langen, Ralf; Stamou, Dimitrios

2011-01-01

Preferential binding of proteins on curved membranes (membrane curvature sensing) is increasingly emerging as a general mechanism whereby cells may effect protein localization and trafficking. Here we use a novel single liposome fluorescence microscopy assay to examine a common sensing motif, the amphipathic helix (AH), and provide quantitative measures describing and distinguishing membrane binding and sensing behavior. By studying two AH-containing proteins, α-synuclein and annexin B12, as well as a range of AH peptide mutants, we reveal that both the hydrophobic and hydrophilic faces of the helix greatly influence binding and sensing. Although increased hydrophobic and electrostatic interactions with the membrane both lead to greater densities of bound protein, the former yields membrane curvature-sensitive binding, whereas the latter is not curvature-dependent. However, the relative contributions of both components determine the sensing of AHs. In contrast, charge density in the lipid membrane seems important primarily in attracting AHs to the membrane but does not significantly influence sensing. These observations were made possible by the ability of our assay to distinguish within our samples liposomes with and without bound protein as well as the density of bound protein. Our findings suggest that the description of membrane curvature-sensing requires consideration of several factors such as short and long range electrostatic interactions, hydrogen bonding, and the volume and structure of inserted hydrophobic residues. PMID:21953452

Progesterone, Inflammatory Cytokine (TNF-α), and Oxidative Stress (H2O2) Regulate Progesterone Receptor Membrane Component 1 Expression in Fetal Membrane Cells.

PubMed

Meng, Yan; Murtha, Amy P; Feng, Liping

2016-09-01

Progesterone receptor membrane component 1 (PGRMC1) is an important novel mediator of progesterone (P4) function in fetal membrane cells. We demonstrated previously that PGRMC1 is differentially expressed in fetal membranes among pregnancy subjects and diminished in preterm premature rupture of membrane subjects. In the current study, we aim to elucidate whether PGRMC1 expression is regulated by P4, tumor necrosis factor α (TNF-α), and H2O2 in fetal membrane cells. Primary cultured membrane cells were serum starved for 24 hours followed by treatments of P4, 17 hydroxyprogesterone caproate, and medroxyprogesterone 17 acetate (MPA) at 10(-7) mol/L with ethanol as vehicle control; TNF-α at 10, 20, and 50 ng/mL with phosphate-buffered saline (PBS) as control; and H2O2 at 10 and 100 μmol/L with culture media as control for 24, 48, and 72 hours. The messenger RNA (mRNA) and protein expression of PGRMC1 was quantified using polymerase chain reaction and Western blotting, respectively. We found that PGRMC1 protein expression was regulated by MPA, TNF-α, and H2O2 in a dose-dependent manner. This regulation is also specific to the type of cell (amnion, chorion, or decidua). The upregulation of PGRMC1 by MPA might be mediated through glucocorticoid receptor (GR) demonstrated using amnion and chorion cells model with GR knockdown by specific small interfering RNA transfection. The mRNA expression of PGRMC1 was decreased by H2O2 (100 μmol/L) treatment in amnion cells, which might ultimately result in downregulation of PGRMC1 protein as our data demonstrated. None of other treatments changed PGRMC1 mRNA level in these cells. We conclude that these stimuli act as regulatory factors of PGRMC1 in a cell-specific manner. © The Author(s) 2016.

Sensing small molecule interactions with lipid membranes by local pH modulation.

PubMed

Huang, Da; Zhao, Tao; Xu, Wei; Yang, Tinglu; Cremer, Paul S

2013-11-05

Herein, we utilized a label-free sensing platform based on pH modulation to detect the interactions between tetracaine, a positively charged small molecule used as a local anesthetic, and planar supported lipid bilayers (SLBs). The SLBs were patterned inside a flow cell, allowing for various concentrations of tetracaine to be introduced over the surface in a buffer solution. Studies with membranes containing POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) yielded an equilibrium dissociation constant value of Kd = 180 ± 47 μm for this small molecule-membrane interaction. Adding cholesterol to the SLBs decreased the affinity between tetracaine and the bilayers, while this interaction tightened when POPE (1-hexadecanoyl-2-(9-Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine) was added. Studies were also conducted with three negatively charged membrane lipids, POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)), POPS (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (sodium salt)), and ganglioside GM1. All three measurements gave rise to a similar tightening of the apparent Kd value compared with pure POPC membranes. The lack of chemical specificity with the identity of the negatively charged lipid indicated that the tightening was largely electrostatic. Through a direct comparison with ITC measurements, it was found that the pH modulation sensor platform offers a facile, inexpensive, highly sensitive, and rapid method for the detection of interactions between putative drug candidates and lipid bilayers. As such, this technique may potentially be exploited as a screen for drug development and analysis.

Process for restoring membrane permeation properties

DOEpatents

Pinnau, Ingo; Toy, Lora G.; Casillas, Carlos G.

1997-05-20

A process for restoring the selectivity of high-flee-volume, glassy polymer membranes for condensable components over less-condensable components or non-condensable components of a gas mixture. The process involves exposing the membrane to suitable sorbent vapor, such as propane or butane, thereby reopening the microvoids that make up the free volume. The selectivity of an aged membrane may be restored to 70-100% of its original value. The selectivity of a membrane which is known to age over time can also be maintained by keeping the membrane in a vapor environment when it is not in use.

Process for restoring membrane permeation properties

DOEpatents

Pinnau, I.; Toy, L.G.; Casillas, C.G.

1997-05-20

A process is described for restoring the selectivity of high-free-volume, glassy polymer membranes for condensable components over less-condensable components or non-condensable components of a gas mixture. The process involves exposing the membrane to suitable sorbent vapor, such as propane or butane, thereby reopening the microvoids that make up the free volume. The selectivity of an aged membrane may be restored to 70--100% of its original value. The selectivity of a membrane which is known to age over time can also be maintained by keeping the membrane in a vapor environment when it is not in use. 8 figs.

Integrin-like proteins are localized to plasma membrane fractions, not plastids, in Arabidopsis

NASA Technical Reports Server (NTRS)

Swatzell, L. J.; Edelmann, R. E.; Makaroff, C. A.; Kiss, J. Z.

1999-01-01

Integrins are a large family of integral membrane proteins that function in signal transduction in animal systems. These proteins are conserved in vertebrates, invertebrates, and fungi. Evidence from previous research suggests that integrin-like proteins may be present in plants as well, and that these proteins may function in signal transduction during gravitropism. In past studies, researchers have used monoclonal and polyclonal antibodies to localize beta 1 integrin-like proteins in plants. However, there is a disparity between data collected from these studies, especially since molecular weights obtained from these investigations range from 55-120 kDa for integrin-like proteins. To date, a complete investigation which employs all three basic immunolabeling procedures, immunoblotting, immunofluorescence microscopy, and immunogold labeling, in addition to extensive fractionation and exhaustive controls, has been lacking. In this paper, we demonstrate that use of a polyclonal antibody against the cytoplasmic domain of avian beta 1-integrin can produce potential artifacts in immunolocalization studies. However, these problems can be eliminated through use of starchless mutants or proper specimen preparation prior to electrophoresis. We also show that this antibody, when applied within the described parameters and with careful controls, identifies a large (100 kDa) integrin-like protein that is localized to plasma membrane fractions in Arabidopsis.

A Local Learning Rule for Independent Component Analysis

PubMed Central

Isomura, Takuya; Toyoizumi, Taro

2016-01-01

Humans can separately recognize independent sources when they sense their superposition. This decomposition is mathematically formulated as independent component analysis (ICA). While a few biologically plausible learning rules, so-called local learning rules, have been proposed to achieve ICA, their performance varies depending on the parameters characterizing the mixed signals. Here, we propose a new learning rule that is both easy to implement and reliable. Both mathematical and numerical analyses confirm that the proposed rule outperforms other local learning rules over a wide range of parameters. Notably, unlike other rules, the proposed rule can separate independent sources without any preprocessing, even if the number of sources is unknown. The successful performance of the proposed rule is then demonstrated using natural images and movies. We discuss the implications of this finding for our understanding of neuronal information processing and its promising applications to neuromorphic engineering. PMID:27323661

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

PubMed

Kress, Alla; Wang, Xiao; Ranchon, Hubert; Savatier, Julien; Rigneault, Hervé; Ferrand, Patrick; Brasselet, Sophie

2013-07-02

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Immunological properties of Micrococcus lysodeikticus membranes.

PubMed

Fukui, Y; Nachbar, M S; Salton, M R

1971-01-01

Membranes of Micrococcus lysodeikticus possess antigens which are distinct from other cellular components such as cytoplasm, ribosomes, and cell walls. Only a few (two to three) components are found when dissociated membranes are examined by immunodiffusion and immunoelectrophoresis techniques. Membranes treated with 0.3% sodium dodecyl sulfate, 0.3% Triton X-100, trypsin, phospholipase A or C, or by sonic oscillation at pH 9.0, all showed the same pattern (three major bands) when examined against membrane antisera by immunoelectrophoresis. Immunological analysis of fractions isolated by sucrose gradient centrifugation or by polyacrylamide gel electrophoresis suggests that individual components cross-react. Antibodies to adenosine triphosphatase (EC 3.6.1.3) and fast-moving component are not removed by absorption with protoplasts. Removal of antibody to one of the membrane antigens by protoplast absorption indicated a surface location. Glutaraldehyde fixation of protoplasts resulted in the loss of membrane antigens detectable by immunodiffusion.

At the border: the plasma membrane-cell wall continuum.

PubMed

Liu, Zengyu; Persson, Staffan; Sánchez-Rodríguez, Clara

2015-03-01

Plant cells rely on their cell walls for directed growth and environmental adaptation. Synthesis and remodelling of the cell walls are membrane-related processes. During cell growth and exposure to external stimuli, there is a constant exchange of lipids, proteins, and other cell wall components between the cytosol and the plasma membrane/apoplast. This exchange of material and the localization of cell wall proteins at certain spots in the plasma membrane seem to rely on a particular membrane composition. In addition, sensors at the plasma membrane detect changes in the cell wall architecture, and activate cytoplasmic signalling schemes and ultimately cell wall remodelling. The apoplastic polysaccharide matrix is, on the other hand, crucial for preventing proteins diffusing uncontrollably in the membrane. Therefore, the cell wall-plasma membrane link is essential for plant development and responses to external stimuli. This review focuses on the relationship between the cell wall and plasma membrane, and its importance for plant tissue organization. © 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.

Localization of extracellular matrix components in developing mouse salivary glands by confocal microscopy

NASA Technical Reports Server (NTRS)

Hardman, P.; Spooner, B. S.

1992-01-01

The importance of the extracellular matrix (ECM) in epithelial-mesenchymal interactions in developing organisms is well established. Proteoglycans and interstitial collagens are required for the growth, morphogenesis, and differentiation of epithelial organs and the distribution of these molecules has been described. However, much less is known about other ECM macromolecules in developing epithelial organs. We used confocal microscopy to examine the distribution of laminin, heparan sulfate (BM-1) proteoglycan, fibronectin, and collagen types I, IV, and V, in mouse embryonic salivary glands. Organ rudiments were isolated from gestational day 13 mouse embryos and cultured for 24, 48, or 72 hours. Whole mounts were stained by indirect immunofluorescence and then examined using a Zeiss Laser Scan Microscope. We found that each ECM component examined had a distinct distribution and that the distribution of some molecules varied with culture time. Laminin was mainly restricted to the basement membrane. BM-1 proteoglycan was concentrated in the basement membrane and also formed a fine network throughout the mesenchyme. Type IV collagen was mainly located in the basement membrane of the epithelium, but it was also present throughout the mesenchyme. Type V collagen was distributed throughout the mesenchyme at 24 hours, but at 48 hours was principally located in the basement membrane. Type I collagen was distributed throughout the mesenchyme at all culture times, and accumulated in the clefts and particularly at the epithelial-mesenchymal interface as time in culture increased. Fibronectin was observed throughout the mesenchyme at all times.

Multiple sound source localization using gammatone auditory filtering and direct sound componence detection

NASA Astrophysics Data System (ADS)

Chen, Huaiyu; Cao, Li

2017-06-01

In order to research multiple sound source localization with room reverberation and background noise, we analyze the shortcomings of traditional broadband MUSIC and ordinary auditory filtering based broadband MUSIC method, then a new broadband MUSIC algorithm with gammatone auditory filtering of frequency component selection control and detection of ascending segment of direct sound componence is proposed. The proposed algorithm controls frequency component within the interested frequency band in multichannel bandpass filter stage. Detecting the direct sound componence of the sound source for suppressing room reverberation interference is also proposed, whose merits are fast calculation and avoiding using more complex de-reverberation processing algorithm. Besides, the pseudo-spectrum of different frequency channels is weighted by their maximum amplitude for every speech frame. Through the simulation and real room reverberation environment experiments, the proposed method has good performance. Dynamic multiple sound source localization experimental results indicate that the average absolute error of azimuth estimated by the proposed algorithm is less and the histogram result has higher angle resolution.

How synthetic membrane systems contribute to the understanding of lipid-driven endocytosis.

PubMed

Schubert, Thomas; Römer, Winfried

2015-11-01

Synthetic membrane systems, such as giant unilamellar vesicles and solid supported lipid bilayers, have widened our understanding of biological processes occurring at or through membranes. Artificial systems are particularly suited to study the inherent properties of membranes with regard to their components and characteristics. This review critically reflects the emerging molecular mechanism of lipid-driven endocytosis and the impact of model membrane systems in elucidating the complex interplay of biomolecules within this process. Lipid receptor clustering induced by binding of several toxins, viruses and bacteria to the plasma membrane leads to local membrane bending and formation of tubular membrane invaginations. Here, lipid shape, and protein structure and valency are the essential parameters in membrane deformation. Combining observations of complex cellular processes and their reconstitution on minimal systems seems to be a promising future approach to resolve basic underlying mechanisms. This article is part of a Special Issue entitled: Mechanobiology. Copyright © 2015 Elsevier B.V. All rights reserved.

Continuity of monolayer-bilayer junctions for localization of lipid raft microdomains in model membranes

DOE PAGES

Ryu, Yong -Sang; Wittenberg, Nathan J.; Suh, Jeng -Hun; ...

2016-05-27

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

Cytochrome b 6 f function and localization, phosphorylation state of thylakoid membrane proteins and consequences on cyclic electron flow.

PubMed

Dumas, Louis; Chazaux, Marie; Peltier, Gilles; Johnson, Xenie; Alric, Jean

2016-09-01

Both the structure and the protein composition of thylakoid membranes have an impact on light harvesting and electron transfer in the photosynthetic chain. Thylakoid membranes form stacks and lamellae where photosystem II and photosystem I localize, respectively. Light-harvesting complexes II can be associated to either PSII or PSI depending on the redox state of the plastoquinone pool, and their distribution is governed by state transitions. Upon state transitions, the thylakoid ultrastructure and lateral distribution of proteins along the membrane are subject to significant rearrangements. In addition, quinone diffusion is limited to membrane microdomains and the cytochrome b 6 f complex localizes either to PSII-containing grana stacks or PSI-containing stroma lamellae. Here, we discuss possible similarities or differences between green algae and C3 plants on the functional consequences of such heterogeneities in the photosynthetic electron transport chain and propose a model in which quinones, accepting electrons either from PSII (linear flow) or NDH/PGR pathways (cyclic flow), represent a crucial control point. Our aim is to give an integrated description of these processes and discuss their potential roles in the balance between linear and cyclic electron flows.

Glasslike Membrane Protein Diffusion in a Crowded Membrane.

PubMed

Munguira, Ignacio; Casuso, Ignacio; Takahashi, Hirohide; Rico, Felix; Miyagi, Atsushi; Chami, Mohamed; Scheuring, Simon

2016-02-23

Many functions of the plasma membrane depend critically on its structure and dynamics. Observation of anomalous diffusion in vivo and in vitro using fluorescence microscopy and single particle tracking has advanced our concept of the membrane from a homogeneous fluid bilayer with freely diffusing proteins to a highly organized crowded and clustered mosaic of lipids and proteins. Unfortunately, anomalous diffusion could not be related to local molecular details given the lack of direct and unlabeled molecular observation capabilities. Here, we use high-speed atomic force microscopy and a novel analysis methodology to analyze the pore forming protein lysenin in a highly crowded environment and document coexistence of several diffusion regimes within one membrane. We show the formation of local glassy phases, where proteins are trapped in neighbor-formed cages for time scales up to 10 s, which had not been previously experimentally reported for biological membranes. Furthermore, around solid-like patches and immobile molecules a slower glass phase is detected leading to protein trapping and creating a perimeter of decreased membrane diffusion.

Mapping of the Localization of Type 1 Angiotensin Receptor in Membrane Microdomains Using Bioluminescence Resonance Energy Transfer-based Sensors*

PubMed Central

Balla, András; Tóth, Dániel J.; Soltész-Katona, Eszter; Szakadáti, Gyöngyi; Erdélyi, László Sándor; Várnai, Péter; Hunyady, László

2012-01-01

Initiation and termination of signaling of the type I angiotensin receptor (AT1-R) can lead to dynamic changes in its localization in plasma membrane microdomains. Several markers were recently developed to investigate membrane microdomains. Here, we used several YFP-labeled fusion constructs (i.e. raft or non-raft plasma membrane markers) to analyze the agonist-induced changes in compartmentalization of AT1-R, including internalization or lateral movement between plasma membrane compartments in response to stimulation using bioluminescence resonance energy transfer measurements. Our data demonstrate that angiotensin II (AngII) stimulus changes the microdomain localization of wild type or mutated (DRY → AAY or TSTS → AAAA) AT1-Rs co-expressed with the fluorescent probes in HEK293 cells. The comparison of the trafficking of AT1-R upon AngII stimulus with those of [Sar1,Ile8]AngII or [Sar1,Ile4,Ile8]AngII stimulus revealed different types of changes, depending on the nature of the ligand. The observed changes in receptor compartmentalization of the AT1-R are strikingly different from those of 5HT-2C and EGF receptors, which demonstrate the usefulness of the bioluminescence resonance energy transfer-based measurements in the investigation of receptor trafficking in the plasma membrane in living cell experiments. PMID:22291018

Using cell membrane chromatography and HPLC-TOF/MS method for in vivo study of active components from roots of Aconitum carmichaeli

PubMed Central

Cao, Yan; Chen, Xiao-Fei; Lü, Di-Ya; Dong, Xin; Zhang, Guo-Qing; Chai, Yi-Feng

2012-01-01

An offline two-dimensional system combining a rat cardiac muscle cell membrane chromatography time-of-flight mass spectrometry (CMC-TOF/MS) with a high Performance liquid chromatography time-of-flight mass spectrometry (HPLC-TOF/MS) was established for investigating the parent components and metabolites in rat urine samples after administration of the roots of Aconitum carmichaeli. On the basis ofthe analysis of the first dimension, retention components of the urine sample were collected into 30 fractions (one fraction per minute). Then offline analysis of the second dimension was carried out. 34 compounds including 24 parent alkaloids and 10 potential metabolites were identified from the dosed rat urine, and then binding affinities of different compounds on cell membranes were compared and influences of some functional groups on activity were estimated with the semi-quantification and curve fitting method. As a result, binding affinities decreased along with the process of deacylation, debenzoylation and demethylation, which may be related to the alleviation of toxicity in the procedure of herb processing or metabolism. Moreover, some minor components in rat urine (Songorine, 14-benzoylneoline, Deoxyaconitine, etc.) exerted relatively strong affinity on cell membranes are worth exploring. The results delivered by the System suggest that the CMC can be applied to in vivo study. PMID:29403691

Membrane Localization is Critical for Activation of the PICK1 BAR Domain

PubMed Central

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

2013-01-01

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

Isolation and properties of mesosomal membrane fractions from Micrococcus lysodeikticus

PubMed Central

Owen, Peter; Freer, John H.

1972-01-01

1. A method is described for the isolation of pure mesosomal membrane fractions from Micrococcus lysodeikticus. 2. Plasmolysis of cells, before wall digestion, was necessary for effective mesosome release. 3. The effect of mild shearing forces, temperature and time upon the release of mesosomal membrane from protoplasts was investigated. 4. The optimum yield of mesosomal membranes from stable protoplasts was achieved at 10mm-Mg2+. 5. Mesosomal membrane vesicle fractions prepared at differing Mg2+ concentrations above 10mm were similar in chemical composition. 6. Comparison of the properties of peripheral and mesosomal membrane fractions revealed major differences in the distribution of protein components, membrane phosphorus, mannose and dehydrogenase activities between the two fractions. 7. Only cytochrome b556 was detected in mesosomal membranes, whereas peripheral membranes contained a full complement of cytochromes. 8. Preliminary investigations suggested the localization of an autolytic enzyme(s) in the mesosomal vesicles. 9. The anatomy of mesosomal and peripheral membrane have been compared by the negative-staining and freeze-fracture technique. 10. The results are discussed in relation to a plausible role for the mesosome. ImagesPLATE 1PLATE 2PLATE 3PLATE 4 PMID:4655825

Water Membrane Evaporator

NASA Technical Reports Server (NTRS)

Ungar, Eugene K.; Almlie, Jay C.

2010-01-01

A water membrane evaporator (WME) has been conceived and tested as an alternative to the contamination-sensitive and corrosion-prone evaporators currently used for dissipating heat from space vehicles. The WME consists mainly of the following components: An outer stainless-steel screen that provides structural support for the components mentioned next; Inside and in contact with the stainless-steel screen, a hydrophobic membrane that is permeable to water vapor; Inside and in contact with the hydrophobic membrane, a hydrophilic membrane that transports the liquid feedwater to the inner surface of the hydrophobic membrane; Inside and in contact with the hydrophilic membrane, an annular array of tubes through which flows the spacecraft coolant carrying the heat to be dissipated; and An inner exclusion tube that limits the volume of feedwater in the WME. In operation, a pressurized feedwater reservoir is connected to the volume between the exclusion tube and the coolant tubes. Feedwater fills the volume, saturates the hydrophilic membrane, and is retained by the hydrophobic membrane. The outside of the WME is exposed to space vacuum. Heat from the spacecraft coolant is conducted through the tube walls and the water-saturated hydrophilic membrane to the liquid/vapor interface at the hydrophobic membrane, causing water to evaporate to space. Makeup water flows into the hydrophilic membrane through gaps between the coolant tubes.

Chronic alcohol exposure affects the cell components involved in membrane traffic in neuronal dendrites.

PubMed

Romero, Ana M; Renau-Piqueras, Jaime; Marín, M Pilar; Esteban-Pretel, Guillermo

2015-01-01

The specific traffic of the membrane components in neurons is a major requirement to establish and maintain neuronal domains-the axonal and the somatodendritic domains-and their polarized morphology. Unlike axons, dendrites contain membranous organelles, which are involved in the secretory pathway, including the endoplasmic reticulum, the Golgi apparatus and post-Golgi apparatus carriers, the cytoskeleton, and plasma membrane. A variety of molecules and factors are also involved in this process. Previous studies have shown that chronic alcohol exposure negatively affects several of these cell components, such as the Golgi apparatus or cytoskeleton in neurons. Yet very little information is available on the possible effects of this exposure on the remaining cell elements involved in intracellular trafficking in neurons, particularly in dendrites. By qualitative and quantitative electron microscopy, immunofluorescence and immunoblotting, we herein show that chronic exposure to moderate levels (30 mM) of ethanol in cultured neurons reduces the volume and surface density of the rough endoplasmic reticulum, and increases the levels of GRP78, a chaperone involved in endoplasmic reticulum stress. Ethanol also significantly diminishes the proportion of neurons that show an extension of Golgi into dendrites and dendritic Golgi outposts, a structure present exclusively in longer, thicker apical dendrites. Both Golgi apparatus types were also fragmented into a large number of cells. We also investigated the effect of alcohol on the levels of microtubule-based motor proteins KIF5, KIF17, KIFC2, dynein, and myosin IIb, responsible for transporting different cargoes in dendrites. Of these, alcohol differently affects several of them by lowering dynein and raising KIF5, KIFC2, and myosin IIb. These results, together with other previously published ones, suggest that practically all the protein trafficking steps in dendrites are altered to a greater or lesser extent by chronic

Morphological study on permeating efficiency and localization of FCLA and HpD through membrane of lung cancer cell

NASA Astrophysics Data System (ADS)

Wu, Yunxia; Xing, Da; Tang, Yonghong

2004-07-01

It is reported that apoptosis of cancer cells in photodynamic therapy (PDT) is caused by 1O2 generated in photosensitization. In order to study the mechanism of this kind of 1O2-induced apoptosis, it is necessary to establish a special technique to dynamically detect intracellular production and localization of 1O2. FCLA, as a chemiluminescence probe to detect singlet oxygen (1O2) and superoxide (O2-.), has been used successfully in photodynamic and sonodynamic diagnosis in tissue level, recently. This paper reported a preliminary result of morphological study on permeating efficiency and localization of FCLA and hematoporphyrin derivative (HpD) through cellular membrane. Human lung cancer cell line (ASTC-a-1) was used in the experiment. The result of this research showed that both HpD and FCLA could permeate through cellular membrane and localize to prinuclear area, when HpD or FCLA was incubated with cells. Although the molecular weight of HpD is close to FCLA's, the permeating efficiency of HpD through membrane was different from that of FCLA. Intracellular FCLA concentration reached a peak after incubation for only 30 - 45 minutes, but amount of HpD in cells approached the equilibrium after incubation for near 22 h. In the experiment, we did not observe the evidence of FCLA or HpD penetrating into nucleolus. This study suggests that it is possibly to use a specific chemiluminescence probe to dynamcially detect the production and localization of 1O2 or 02-. in cell.

Developmental Localization of Nephrin in Zebrafish and Medaka Pronephric Glomerulus

PubMed Central

Ichimura, Koichiro; Fukuyo, Yayoi; Nakamura, Tomomi; Powell, Rebecca; Sakai, Tatsuo; Janknecht, Ralf

2013-01-01

Slit diaphragm (SD) is a highly specialized intercellular junction between podocyte foot processes and plays a crucial role in the formation of the filtration barrier. In this study, we examined the developmental localization of Nephrin, an essential component of SD, in the pronephric glomerulus of zebrafish and medaka. In the mature glomerulus of both fish, Nephrin is localized along the glomerular basement membrane as seen in mammals, indicating that Nephrin is localized at the SD. Interestingly, Nephrin was detected already in immature podocytes before the SD and foot processes started to form in both fish. Nephrin was localized along the cell surface of immature podocytes but as different localization patterns. In zebrafish, Nephrin signal bordered the lateral membrane of podocytes, which were columnar in shape, as in rat immature podocytes. However, in medaka immature podocytes, Nephrin was localized in a punctate pattern among podocyte cell bodies. These findings suggest that Nephrin needs to be integrated to the membrane before the formation of the SD and then moves to the proper site to form the SD. Furthermore, a podocyte-specific marker, such as Nephrin, should be a useful tool for the future analysis of pronephric glomerular development in fish mutants and morphants. PMID:23324868

A Novel Di-Leucine Motif at the N-Terminus of Human Organic Solute Transporter Beta Is Essential for Protein Association and Membrane Localization.

PubMed

Xu, Shuhua; Soroka, Carol J; Sun, An-Qiang; Backos, Donald S; Mennone, Albert; Suchy, Frederick J; Boyer, James L

2016-01-01

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

PubMed

Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

2007-08-17

Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 < 1 min) between the plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

Modeling Membrane Deformations and Lipid Demixing upon Protein-Membrane Interaction: The BAR Dimer Adsorption

PubMed Central

Khelashvili, George; Harries, Daniel; Weinstein, Harel

2009-01-01

We use a self-consistent mean-field theory, designed to investigate membrane reshaping and lipid demixing upon interaction with proteins, to explore BAR domains interacting with large patches of lipid membranes of heterogeneous compositions. The computational model includes contributions to the system free energy from electrostatic interactions and elastic energies of the membrane, as well as salt and lipid mixing entropies. The results from our simulation of a single adsorbing Amphiphysin BAR dimer indicate that it is capable of stabilizing a significantly curved membrane. However, we predict that such deformations will occur only for membrane patches that have the inherent propensity for high curvature, reflected in the tendency to create local distortions that closely match the curvature of the BAR dimer itself. Such favorable preconditioning for BAR-membrane interaction may be the result of perturbations such as local lipid demixing induced by the interaction, or of a prior insertion of the BAR domain's amphiphatic N-helix. From our simulations it appears that local segregation of charged lipids under the influence of the BAR dimer cannot produce high enough asymmetry between bilayer leaflets to induce significant bending. In the absence of additional energy contributions that favor membrane asymmetry, the membrane will remain nearly flat upon single BAR dimer adsorption, relative to the undulation expected from thermal fluctuations. Thus, we conclude that the N-helix insertions have a critical mechanistic role in the local perturbation and curving of the membrane, which is then stabilized by the electrostatic interaction with the BAR dimer. We discuss how these results can be used to estimate the tendency of BARs to bend membranes in terms of a spatially nonisotropic spontaneous curvature. PMID:19751667

Probing microscopic material properties inside simulated membranes through spatially resolved three-dimensional local pressure fields and surface tensions

PubMed Central

Kasson, Peter M.; Hess, Berk; Lindahl, Erik

2013-01-01

Cellular lipid membranes are spatially inhomogeneous soft materials. Materials properties such as pressure and surface tension thus show important microscopic-scale variation that is critical to many biological functions. We present a means to calculate pressure and surface tension in a 3D-resolved manner within molecular-dynamics simulations and show how such measurements can yield important insight. We also present the first corrections to local virial and pressure fields to account for the constraints typically used in lipid simulations that otherwise cause problems in highly oriented systems such as bilayers. Based on simulations of an asymmetric bacterial ion channel in a POPC bilayer, we demonstrate how 3D-resolved pressure can probe for both short-range and long-range effects from the protein on the membrane environment. We also show how surface tension is a sensitive metric for inter-leaflet equilibrium and can be used to detect even subtle imbalances between bilayer leaflets in a membrane-protein simulation. Since surface tension is known to modulate the function of many proteins, this effect is an important consideration for predictions of ion channel function. We outline a strategy by which our local pressure measurements, which we make available within a version of the GROMACS simulation package, may be used to design optimally equilibrated membrane-protein simulations. PMID:23318532

Polymeric and Lipid Membranes-From Spheres to Flat Membranes and vice versa.

PubMed

Saveleva, Mariia S; Lengert, Ekaterina V; Gorin, Dmitry A; Parakhonskiy, Bogdan V; Skirtach, Andre G

2017-08-15

Membranes are important components in a number of systems, where separation and control of the flow of molecules is desirable. Controllable membranes represent an even more coveted and desirable entity and their development is considered to be the next step of development. Typically, membranes are considered on flat surfaces, but spherical capsules possess a perfect "infinite" or fully suspended membranes. Similarities and transitions between spherical and flat membranes are discussed, while applications of membranes are also emphasized.

Comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatography for screening anti-tumor components from Radix Sophorae flavescentis.

PubMed

Wang, Qiang; Xu, Junnan; Li, Xiang; Zhang, Dawei; Han, Yong; Zhang, Xu

2017-07-01

Radix Sophorae flavescentis is generally used for the treatment of different stages of prostate cancer in China. It has ideal effects when combined with surgical treatment and chemotherapy. However, its active components are still ambiguous. We devised a comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatography system for screening anti-prostate cancer components in Radix Sophorae flavescentis. Gefitinib and dexamethasone were chosen as positive and negative drugs respectively for validation and optimization the selectivity and suitability of the comprehensive two-dimensional chromatographic system. Five compounds, sophocarpine, matrine, oxymatrine, oxysophocarpine, and xanthohumol were found to have significant retention behaviors on the PC-3 cell membrane chromatography and were unambiguously identified by time-of-flight mass spectrometry. Cell proliferation and apoptosis assays confirmed that all five compounds had anti-prostate cancer effects. Matrine and xanthohumol had good inhibitory effects, with half maximal inhibitory concentration values of 0.893 and 0.137 mg/mL, respectively. Our comprehensive two-dimensional PC-3 prostate cancer cell membrane chromatographic system promotes the efficient recognition and rapid analysis of drug candidates, and it will be practical for the discovery of prostate cancer drugs from complex traditional Chinese medicines. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamic FtsA and FtsZ localization and outer membrane alterations during polar growth and cell division in Agrobacterium tumefaciens

PubMed Central

Zupan, John R.; Cameron, Todd A.; Anderson-Furgeson, James; Zambryski, Patricia C.

2013-01-01

Growth and cell division in rod-shaped bacteria have been primarily studied in species that grow predominantly by peptidoglycan (PG) synthesis along the length of the cell. Rhizobiales species, however, predominantly grow by PG synthesis at a single pole. Here we characterize the dynamic localization of several Agrobacterium tumefaciens components during the cell cycle. First, the lipophilic dye FM 4-64 predominantly stains the outer membranes of old poles versus growing poles. In cells about to divide, however, both poles are equally labeled with FM 4-64, but the constriction site is not. Second, the cell-division protein FtsA alternates from unipolar foci in the shortest cells to unipolar and midcell localization in cells of intermediate length, to strictly midcell localization in the longest cells undergoing septation. Third, the cell division protein FtsZ localizes in a cell-cycle pattern similar to, but more complex than, FtsA. Finally, because PG synthesis is spatially and temporally regulated during the cell cycle, we treated cells with sublethal concentrations of carbenicillin (Cb) to assess the role of penicillin-binding proteins in growth and cell division. Cb-treated cells formed midcell circumferential bulges, suggesting that interrupted PG synthesis destabilizes the septum. Midcell bulges contained bands or foci of FtsA-GFP and FtsZ-GFP and no FM 4-64 label, as in untreated cells. There were no abnormal morphologies at the growth poles in Cb-treated cells, suggesting unipolar growth uses Cb-insensitive PG synthesis enzymes. PMID:23674672

A vitellogenic-like carboxypeptidase expressed by human macrophages is localized in endoplasmic reticulum and membrane ruffles

PubMed Central

Harris, James; Schwinn, Nicole; Mahoney, James A; Lin, Hsi-Hsien; Shaw, Michael; Howard, Chris J; da Silva, Rosangela P; Gordon, Siamon

2006-01-01

Carboxypeptidase, vitellogenic-like (CPVL) is a serine carboxypeptidase of unknown function that was first characterized in human macrophages. Initial studies suggested that CPVL is largely restricted to the monocytic lineage, although it may also be expressed by cells outside the immune system. Here, we use a new monoclonal antibody to characterize the properties and localization of CPVL in human macrophages to elucidate a possible function for the protease. CPVL is up-regulated during the maturation of monocytes (MO) to macrophages, although the protein can be seen in both. In primary macrophages, CPVL is glycosylated with high mannose residues and colocalizes with markers for endoplasmic reticulum, while in MO it is more disperse and less clearly associated with endoplasmic reticulum. CPVL is highly expressed in lamellipodia and membrane ruffles, which also concentrate markers of the secretory pathway (MIP-1α and tumour necrosis factor-α) and major histocompatibility complex (MHC) class I and II molecules. CPVL can be seen on early latex bead and Candida albicans phagosomes, but it is not retained in the maturing phagosome, unlike MHC class I/II. CPVL has a mixed cytosolic and membrane-associated localization but is not detectable on the outer plasma membrane. We propose that CPVL may be involved in antigen processing, the secretory pathway and/or in actin remodelling and lamellipodium formation. PMID:16436111

Multicomponent membranes

DOEpatents

Kulprathipanja, Santi; Kulkarni, Sudhir S.; Funk, Edward W.

1988-01-01

A multicomponent membrane which may be used for separating various components which are present in a fluid feed mixture comprises a mixture of a plasticizer such as a glycol and an organic polymer cast upon a porous organic polymer support. The membrane may be prepared by casting an emulsion or a solution of the plasticizer and polymer on the porous support, evaporating the solvent and recovering the membrane after curing.

Functional link between plasma membrane spatiotemporal dynamics, cancer biology, and dietary membrane-altering agents.

PubMed

Erazo-Oliveras, Alfredo; Fuentes, Natividad R; Wright, Rachel C; Chapkin, Robert S

2018-06-02

The cell plasma membrane serves as a nexus integrating extra- and intracellular components, which together enable many of the fundamental cellular signaling processes that sustain life. In order to perform this key function, plasma membrane components assemble into well-defined domains exhibiting distinct biochemical and biophysical properties that modulate various signaling events. Dysregulation of these highly dynamic membrane domains can promote oncogenic signaling. Recently, it has been demonstrated that select membrane-targeted dietary bioactives (MTDBs) have the ability to remodel plasma membrane domains and subsequently reduce cancer risk. In this review, we focus on the importance of plasma membrane domain structural and signaling functionalities as well as how loss of membrane homeostasis can drive aberrant signaling. Additionally, we discuss the intricacies associated with the investigation of these membrane domain features and their associations with cancer biology. Lastly, we describe the current literature focusing on MTDBs, including mechanisms of chemoprevention and therapeutics in order to establish a functional link between these membrane-altering biomolecules, tuning of plasma membrane hierarchal organization, and their implications in cancer prevention.

CURVATURE-DRIVEN MOLECULAR FLOW ON MEMBRANE SURFACE*

PubMed Central

MIKUCKI, MICHAEL; ZHOU, Y. C.

2017-01-01

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

Intracellular redox status controls membrane localization of pro- and anti-migratory signaling molecules.

PubMed

Hempel, Nadine; Melendez, J Andres

2014-01-01

Shifts in intracellular Reactive Oxygen Species (ROS) have been shown to contribute to carcinogenesis and to tumor progression. In addition to DNA and cell damage by surges in ROS, sub-lethal increases in ROS are implicated in regulating cellular signaling that enhances pro-metastatic behavior. We previously showed that subtle increases in endogenous H2O2 regulate migratory and invasive behavior of metastatic bladder cancer cells through phosphatase inhibition and consequential phosphorylation of p130cas, an adapter of the FAK signaling pathway. We further showed that enhanced redox status contributed to enhanced localization of p130cas to the membrane of metastatic cells. Here we show that this signaling complex can similarly be induced in a redox-engineered cell culture model that enables regulation of intracellular steady state H2O2 level by enforced expression of superoxide dismutase 2 (Sod2) and catalase. Expression of Sod2 leads to enhanced p130cas phosphorylation in HT-1080 fibrosarcoma and UM-UC-6 bladder cancer cells. These changes are mediated by H2O2, as co-expression of Catalase abrogates p130cas phosphorylation and its interaction with the adapter protein Crk. Importantly, we establish that the redox environment influence the localization of the tumor suppressor and phosphatase PTEN, in both redox-engineered and metastatic bladder cancer cells that display endogenous increases in H2O2. Importantly, PTEN oxidation leads to its dissociation from the plasma membrane. This indicates that oxidation of PTEN not only influences its activity, but also regulates its cellular localization, effectively removing it from its primary site of lipid phosphatase activity. These data introduce hitherto unappreciated paradigms whereby ROS can reciprocally regulate the cellular localization of pro- and anti-migratory signaling molecules, p130cas and PTEN, respectively. These data further confirm that altering antioxidant status and the intracellular ROS environment can

The X-ray Emitting Components towards l = 111 deg: The Local Hot Bubble and Beyond

NASA Technical Reports Server (NTRS)

Kuntz, K. D.; Snowden, S. L.

2006-01-01

We have obtained an XMM-Newton spectrum of the diffuse X-ray emission towards (l, b) = (111.14,1.11), a line of sight with a relatively simple distribution of absorbing clouds; > 9 x 10(exp 19)/sq cm at R>170 pc, a 6 x 10(exp 21)/sq cm molecular cloud at 2.5-3.3 kpc, and a total column of 1.2 x 10(exp 22)/sq cm. We find that the analysis of the XMM-Newton spectrum in conjunction with the RASS spectral energy distribution for the same direction requires three thermal components to be well fit: a "standard" Local Hot Bubble component with kT = 0.089, a component beyond the molecular cloud with kT = 0.59, and a component before the molecular cloud with kT = 0.21. The strength of the O VII 0.56 keV line from the Local Hot Bubble, 2.1+/-0.7 photons/sq cm/s/sr, is consistent with other recent measures. The 0.21 keV component has an emission measure of 0.0022+/-0.0006 pc and is not localized save as diffuse emission within the Galactic plane; it is the best candidate for a pervasive hot medium. The spatial separation of the approx. 0.2 keV component from the approx. 0.6 keV component suggests that the spectral decompositions of the emission from late-type spiral disks found in the literature do represent real temperature components rather than reflecting more complex temperature distributions.

Influence of the membrane environment on cholesterol transfer.

PubMed

Breidigan, Jeffrey Michael; Krzyzanowski, Natalie; Liu, Yangmingyue; Porcar, Lionel; Perez-Salas, Ursula

2017-12-01

Cholesterol, an essential component in biological membranes, is highly unevenly distributed within the cell, with most localized in the plasma membrane while only a small fraction is found in the endoplasmic reticulum, where it is synthesized. Cellular membranes differ in lipid composition and protein content, and these differences can exist across their leaflets too. This thermodynamic landscape that cellular membranes impose on cholesterol is expected to modulate its transport. To uncover the role the membrane environment has on cholesterol inter- and intra-membrane movement, we used time-resolved small angle neutron scattering to study the passive movement of cholesterol between and within membranes with varying degrees of saturation content. We found that cholesterol moves systematically slower as the degree of saturation in the membranes increases, from a palmitoyl oleyl phosphotidylcholine membrane, which is unsaturated, to a dipalmitoylphosphatidylcholine (DPPC) membrane, which is fully saturated. Additionally, we found that the energetic barrier to move cholesterol in these phosphatidylcholine membranes is independent of their relative lipid composition and remains constant for both flip-flop and exchange at ∼100 kJ/mol. Further, by replacing DPPC with the saturated lipid palmitoylsphingomyelin, an abundant saturated lipid of the outer leaflet of the plasma membrane, we found the rates decreased by a factor of two. This finding is in stark contrast with recent molecular dynamic simulations that predict a dramatic slow-down of seven orders of magnitude for cholesterol flipping in membranes with a similar phosphocholine and SM lipid composition. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

Radiographic contrast media alterate the localization of actin/band4.9 in the membrane cytoskeleton of human erythrocytes.

PubMed

Franke, R P; Scharnweber, T; Fuhrmann, R; Mrowietz, C; Wenzel, F; Krüger, A; Jung, F

2014-01-01

Different radiographic contrast media (RCM) were shown to induce morphological changes of blood cells (e.g. erythrocytes or thrombocytes) and endothelial cells. The echinocytic shape change of erythrocytes, particularly, affords alterations of the membrane cytoskeleton. The cytoskeleton plays a crucial role for the shape and deformability of the red blood cell. Disruption of the interaction between components of the red blood cell membrane cytoskeleton may cause a loss of structural and functional integrity of the membrane. In this study band4.9 and actin as components of the cytoskeletal junctional complex were examined in human erythrocytes after suspension in autologous plasma or in plasma RCM mixtures (30% v/v Iodixanol-320 or Iopromide-370) followed by a successive double staining with TRITC-/FITC-coupled monoclonal antibodies. After adding Iopromide-370 to the plasma in practically none of the cells the rounded conformation of the membrane cytoskeleton - as it appeared in cells suspended in autologous plasma - was found. In addition, Iopromide-370 induced thin lines and coarse knob-like structures of band4.9 at the cell periphery while most cell centers were devoid of band4.9, and a box-like arrangement of bands of band4.9. A dissociation between colours red (actin) and green (band4.9) occurred as well. In contrast, erythrocytes suspended in a plasma/Iodixanol-320 mixture showed a membrane cytoskeleton comparable to cells suspended in autologous plasma, Similar results were found with respect to the distribution of actin. This study revealed for the first time RCM-dependent differences in band4.9 activities as possible pathophysiological mechanism for the chemotoxicity of radiographic contrast media.

Concentration gradient along the scala tympani after local application of gentamicin to the round window membrane.

PubMed

Plontke, Stefan K; Mynatt, Robert; Gill, Ruth M; Borgmann, Stefan; Salt, Alec N

2007-07-01

The distribution of gentamicin along the fluid spaces of the cochlea after local applications has never previously been demonstrated. Computer simulations have predicted that significant basal-apical concentration gradients might be expected, and histologic studies indicate that hair cell damage is greater at the base than at the apex after local gentamicin application. In the present study, gradients of gentamicin along the cochlea were measured. A recently developed method of sampling perilymph from the cochlear apex of guinea pigs was used in which the samples represent fluid originating from different regions along the scala tympani. Gentamicin concentration was determined in sequential apical samples that were taken after up to 3 hours of local application to the round window niche. Substantial gradients of gentamicin along the length of the scala tympani were demonstrated and quantified, averaging more than 4,000 times greater concentration at the base compared with the apex at the time of sampling. Peak concentrations and gradients for gentamicin varied considerably between animals, likely resulting from variations in round window membrane permeability and rates of perilymph flow. The large gradients for gentamicin demonstrated here in guinea pigs account for how it is possible to suppress vestibular function in some patients with a local application of gentamicin without damaging auditory function. Variations in round window membrane permeability and in perilymph flow could account for why hearing losses are observed in some patients.

Concentration gradient along scala tympani following the local application of gentamicin to the round window membrane

PubMed Central

Plontke, Stefan K.; Mynatt, Robert; Gill, Ruth M.; Borgmann, Stefan; Salt, Alec N.

2008-01-01

Objectives The distribution of gentamicin along the fluid spaces of the cochlea following local applications has never previously been demonstrated. Computer simulations have predicted that significant basal-apical concentration gradients might be expected and histological studies indicate that hair cell damage is greater at the base than at the apex following local gentamicin application. In the present study, gradients of gentamicin along the cochlea were measured. Methods A recently-developed method of sampling perilymph from the cochlear apex of guinea pigs was used, in which the samples represent fluid originating from different regions along scala tympani. Gentamicin concentration was determined in sequential apical samples which were taken following up to three hours of local application to the round window niche. Results Substantial gradients of gentamicin along the length of scala tympani were demonstrated and quantified, averaging more than 4000 times greater concentration at the base compared to the apex at the time of sampling. Peak concentrations and gradients for gentamicin varied considerably between animals, likely resulting from variations in round window membrane permeability and rates of perilymph flow. Conclusions The large gradients for gentamicin demonstrated here in guinea pigs account for how it is possible to suppress vestibular function in some patients with a local application of gentamicin without damaging auditory function. Variations in round window membrane permeability and in perilymph flow could account for why hearing losses are observed in some patients. PMID:17603318

Membrane permeation process for dehydration of organic liquid mixtures using sulfonated ion-exchange polyalkene membranes

DOEpatents

Cabasso, Israel; Korngold, Emmanuel

1988-01-01

A membrane permeation process for dehydrating a mixture of organic liquids, such as alcohols or close boiling, heat sensitive mixtures. The process comprises causing a component of the mixture to selectively sorb into one side of sulfonated ion-exchange polyalkene (e.g., polyethylene) membranes and selectively diffuse or flow therethrough, and then desorbing the component into a gas or liquid phase on the other side of the membranes.

Using the Localized Surface Plasmon Resonance of Gold Nanoparticles to Monitor Lipid Membrane Assembly and Protein Binding.

PubMed

Messersmith, Reid E; Nusz, Greg J; Reed, Scott M

2013-12-19

Gold nanoparticles provide a template for preparing supported lipid layers with well-defined curvature. Here, we utilize the localized surface plasmon resonance (LSPR) of gold nanoparticles as a sensor for monitoring the preparation of lipid layers on nanoparticles. The LSPR is very sensitive to the immediate surroundings of the nanoparticle surface and it is used to monitor the coating of lipids and subsequent conversion of a supported bilayer to a hybrid membrane with an outer lipid leaflet and an inner leaflet containing hydrophobic alkanethiol. We demonstrate that both decanethiol and propanethiol are able to form hybrid membranes and that the membrane created over the shorter thiol can be stripped from the gold along with the lipid leaflet using β-mercaptoethanol. The sensitivity of the nanoparticle LSPR to the refractive index (RI) of its surroundings is greater when the shorter thiol is used (37.8 ± 1.5 nm per RI unit) than when the longer thiol is used (27.5 ± 0.5 nm per RI unit). Finally, C-reactive protein binding to the membrane is measured using this sensor allowing observation of both protein-membrane and nanoparticle-nanoparticle interactions without chemical labeling of protein or lipids.

Localization of intracellular and plasma membrane Ca2+-ATPases in the cerebellum.

PubMed

Sepúlveda, M Rosario; Mata, Ana M

2005-01-01

The sarco-endoplasmic reticulum Ca2+-ATPase and the plasma membrane Ca2+-ATPase contribute to the regulation of the intracellular Ca2+ concentration. These proteins transport Ca2+ ions into the endoplasmic reticulum and to the extracellular medium, respectively. A different localization of the two families of Ca2+-ATPases has been shown in concrete subcellular areas of Purkinje cells and in other neuronal elements from cerebellum. In the light of the actual knowledge of Ca2+-ATPases, this strict distribution suggests the existence of different demands on Ca2+ homeostasis in these cerebellar and cellular subregions.

Structural and mechanical heterogeneity of the erythrocyte membrane reveals hallmarks of membrane stability.

PubMed

Picas, Laura; Rico, Félix; Deforet, Maxime; Scheuring, Simon

2013-02-26

The erythrocyte membrane, a metabolically regulated active structure that comprises lipid molecules, junctional complexes, and the spectrin network, enables the cell to undergo large passive deformations when passing through the microvascular system. Here we use atomic force microscopy (AFM) imaging and quantitative mechanical mapping at nanometer resolution to correlate structure and mechanics of key components of the erythrocyte membrane, crucial for cell integrity and function. Our data reveal structural and mechanical heterogeneity modulated by the metabolic state at unprecedented nanometer resolution. ATP-depletion, reducing skeletal junction phosphorylation in RBC cells, leads to membrane stiffening. Analysis of ghosts and shear-force opened erythrocytes show that, in the absence of cytosolic kinases, spectrin phosphorylation results in membrane stiffening at the extracellular face and a reduced junction remodeling in response to loading forces. Topography and mechanical mapping of single components at the cytoplasmic face reveal that, surprisingly, spectrin phosphorylation by ATP softens individual filaments. Our findings suggest that, besides the mechanical signature of each component, the RBC membrane mechanics is regulated by the metabolic state and the assembly of its structural elements.

The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

PubMed

Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

2016-05-17

Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Robust membrane detection based on tensor voting for electron tomography.

PubMed

Martinez-Sanchez, Antonio; Garcia, Inmaculada; Asano, Shoh; Lucic, Vladan; Fernandez, Jose-Jesus

2014-04-01

Electron tomography enables three-dimensional (3D) visualization and analysis of the subcellular architecture at a resolution of a few nanometers. Segmentation of structural components present in 3D images (tomograms) is often necessary for their interpretation. However, it is severely hampered by a number of factors that are inherent to electron tomography (e.g. noise, low contrast, distortion). Thus, there is a need for new and improved computational methods to facilitate this challenging task. In this work, we present a new method for membrane segmentation that is based on anisotropic propagation of the local structural information using the tensor voting algorithm. The local structure at each voxel is then refined according to the information received from other voxels. Because voxels belonging to the same membrane have coherent structural information, the underlying global structure is strengthened. In this way, local information is easily integrated at a global scale to yield segmented structures. This method performs well under low signal-to-noise ratio typically found in tomograms of vitrified samples under cryo-tomography conditions and can bridge gaps present on membranes. The performance of the method is demonstrated by applications to tomograms of different biological samples and by quantitative comparison with standard template matching procedure. Copyright © 2014 Elsevier Inc. All rights reserved.

Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss

PubMed Central

Cizmecioglu, Onur; Ni, Jing; Xie, Shaozhen; Zhao, Jean J; Roberts, Thomas M

2016-01-01

We aimed to understand how spatial compartmentalization in the plasma membrane might contribute to the functions of the ubiquitous class IA phosphoinositide 3-kinase (PI3K) isoforms, p110α and p110β. We found that p110β localizes to membrane rafts in a Rac1-dependent manner. This localization potentiates Akt activation by G-protein-coupled receptors (GPCRs). Thus genetic targeting of a Rac1 binding-deficient allele of p110β to rafts alleviated the requirement for p110β-Rac1 association for GPCR signaling, cell growth and migration. In contrast, p110α, which does not play a physiological role in GPCR signaling, is found to reside in nonraft regions of the plasma membrane. Raft targeting of p110α allowed its EGFR-mediated activation by GPCRs. Notably, p110β dependent, PTEN null tumor cells critically rely upon raft-associated PI3K activity. Collectively, our findings provide a mechanistic account of how membrane raft localization regulates differential activation of distinct PI3K isoforms and offer insight into why PTEN-deficient cancers depend on p110β. DOI: http://dx.doi.org/10.7554/eLife.17635.001 PMID:27700986

Golgi bypass for local delivery of axonal proteins, fact or fiction?

PubMed

González, Carolina; Cornejo, Víctor Hugo; Couve, Andrés

2018-04-06

Although translation of cytosolic proteins is well described in axons, much less is known about the synthesis, processing and trafficking of transmembrane and secreted proteins. A canonical rough endoplasmic reticulum or a stacked Golgi apparatus has not been detected in axons, generating doubts about the functionality of a local route. However, axons contain mRNAs for membrane and secreted proteins, translation factors, ribosomal components, smooth endoplasmic reticulum and post-endoplasmic reticulum elements that may contribute to local biosynthesis and plasma membrane delivery. Here we consider the evidence supporting a local secretory system in axons. We discuss exocytic elements and examples of autonomous axonal trafficking that impact development and maintenance. We also examine whether unconventional post-endoplasmic reticulum pathways may replace the canonical Golgi apparatus. Copyright © 2018. Published by Elsevier Ltd.

Septal membrane localization by C-terminal amphipathic α-helices of MinD in Bacillus subtilis mutant cells lacking MinJ or DivIVA.

PubMed

Ishikawa, Kazuki; Matsuoka, Satoshi; Hara, Hiroshi; Matsumoto, Kouji

2017-10-18

The Min system, which inhibits assembly of the cytokinetic protein FtsZ, is largely responsible for positioning the division site in rod-shaped bacteria. It has been reported that MinJ, which bridges DivIVA and MinD, is targeted to the cell poles by an interaction with DivIVA, and that MinJ in turn recruits MinCD to the cell poles. MinC, however, is located primarily at active division sites at mid-cell when expressed from its native promoter. Surprisingly, we found that Bacillus subtilis MinD is located at nascent septal membranes and at an asymmetric site on lateral membranes between nascent septal membranes in filamentous cells lacking MinJ or DivIVA. Bacillus subtilis MinD has two amphipathic α-helices rich in basic amino acid residues at its C-terminus; one of these, named MTS1 here, is the counterpart of the membrane targeting sequence (MTS) in Escherichia coli MinD while the other, named MTS-like sequence (MTSL), is the nearest helix to MTS1. These amphipathic helices were located independently at nascent septal membranes in cells lacking MinJ or DivIVA, whereas elimination of the helices from the wild type protein reduced its localization considerably. MinD variants with altered MTS1 and MTSL, in which basic amino acid residues were replaced with proline or acidic residues, were not located at nascent septal membranes, indicating that the binding to the nascent septal membranes requires basic residues and a helical structure. The septal localization of MTSL, but not of MTS1, was dependent on host cell MinD. These results suggest that MinD is targeted to nascent septal membranes via its C-terminal amphipathic α-helices in B. subtilis cells lacking MinJ or DivIVA. Moreover, the diffuse distribution of MinD lacking both MTSs suggests that only a small fraction of MinD depends on MinJ for its localization to nascent septal membranes.

Local Laser Strengthening of Steel Sheets for Load Adapted Component Design in Car Body Structures

NASA Astrophysics Data System (ADS)

Jahn, Axel; Heitmanek, Marco; Standfuss, Jens; Brenner, Berndt; Wunderlich, Gerd; Donat, Bernd

The current trend in car body construction concerning light weight design and car safety improvement increasingly requires an adaption of the local material properties on the component load. Martensitic hardenable steels, which are typically used in car body components, show a significant hardening effect, for instance in laser welded seams. This effect can be purposefully used as a local strengthening method. For several steel grades the local strengthening, resulting from a laser remelting process was investigated. The strength in the treated zone was determined at crash relevant strain rates. A load adapted design of complex reinforcement structures was developed for compression and bending loaded tube samples, using numerical simulation of the deformation behavior. Especially for bending loaded parts, the crash energy absorption can be increased significantly by local laser strengthening.

Modeling of Protein Subcellular Localization in Bacteria

NASA Astrophysics Data System (ADS)

Xu, Xiaohua; Kulkarni, Rahul

2006-03-01

Specific subcellular localization of proteins is a vital component of important bacterial processes: e.g. the Min proteins which regulate cell division in E. coli and Spo0J-Soj system which is critical for sporulation in B. subtilis. We examine how the processes of diffusion and membrane attachment contribute to protein subcellular localization for the above systems. We use previous experimental results to suggest minimal models for these processes. For the minimal models, we derive analytic expressions which provide insight into the processes that determine protein subcellular localization. Finally, we present the results of numerical simulations for the systems studied and make connections to the observed experiemental phenomenology.

Impact of heat and water management on proton exchange membrane fuel cells degradation in automotive application

NASA Astrophysics Data System (ADS)

Nandjou, F.; Poirot-Crouvezier, J.-P.; Chandesris, M.; Blachot, J.-F.; Bonnaud, C.; Bultel, Y.

2016-09-01

In Proton Exchange Membrane Fuel Cells, local temperature is a driving force for many degradation mechanisms such as hygrothermal deformation and creep of the membrane, platinum dissolution and bipolar plates corrosion. In order to investigate and quantify those effects in automotive application, durability testing is conducted in this work. During the ageing tests, the local performance and temperature are investigated using in situ measurements of a printed circuit board. At the end of life, post-mortem analyses of the aged components are conducted. The experimental results are compared with the simulated temperature and humidity in the cell obtained from a pseudo-3D multiphysics model in order to correlate the observed degradations to the local conditions inside the stack. The primary cause of failure in automotive cycling is pinhole/crack formation in the membrane, induced by high variations of its water content over time. It is also observed that water condensation largely increases the probability of the bipolar plates corrosion while evaporation phenomena induce local deposits in the cell.

Low-intensity electric fields induce two distinct response components in neocortical neuronal populations

PubMed Central

Xu, Weifeng; Wolff, Brian S.

2014-01-01

Low-intensity alternating electric fields applied to the scalp are capable of modulating cortical activity and brain functions, but the underlying mechanisms remain largely unknown. Here, we report two distinct components of voltage-sensitive dye signals induced by low-intensity, alternating electric fields in rodent cortical slices: a “passive component,” which corresponds to membrane potential changes directly induced by the electric field; and an “active component,” which is a widespread depolarization that is dependent on excitatory synaptic transmission. The passive component is stationary, with amplitude and phase accurately reflecting the cortical cytoarchitecture. In contrast, the active component is initiated from a local “hot spot” of activity and spreads to a large population as a propagating wave with rich local dynamics. The propagation of the active component may play a role in modulating large-scale cortical activity by spreading a low level of excitation from a small initiation point to a vast neuronal population. PMID:25122710

Process for recycling components of a PEM fuel cell membrane electrode assembly

DOEpatents

Shore, Lawrence [Edison, NJ

2012-02-28

The membrane electrode assembly (MEA) of a PEM fuel cell can be recycled by contacting the MEA with a lower alkyl alcohol solvent which separates the membrane from the anode and cathode layers of the assembly. The resulting solution containing both the polymer membrane and supported noble metal catalysts can be heated under mild conditions to disperse the polymer membrane as particles and the supported noble metal catalysts and polymer membrane particles separated by known filtration means.

Vitamin D receptor is present on the neuronal plasma membrane and is co-localized with amyloid precursor protein, ADAM10 or Nicastrin.

PubMed

Dursun, Erdinç; Gezen-Ak, Duygu

2017-01-01

Our recent study indicated that vitamin D and its receptors are important parts of the amyloid processing pathway in neurons. Yet the role of vitamin D receptor (VDR) in amyloid pathogenesis is complex and all regulations over the production of amyloid beta cannot be explained solely with the transcriptional regulatory properties of VDR. Given that we hypothesized that VDR might exist on the neuronal plasma membrane in close proximity with amyloid precursor protein (APP) and secretase complexes. The present study primarily focused on the localization of VDR in neurons and its interaction with amyloid pathology-related proteins. The localization of VDR on neuronal membranes and its co-localization with target proteins were investigated with cell surface staining followed by immunofluorescence labelling. The FpClass was used for protein-protein interaction prediction. Our results demonstrated the localization of VDR on the neuronal plasma membrane and the co-localization of VDR and APP or ADAM10 or Nicastrin and limited co-localization of VDR and PS1. E-cadherin interaction with APP or the γ-secretase complex may involve NOTCH1, NUMB, or FHL2, according to FpClass. This suggested complex might also include VDR, which greatly contributes to Ca+2 hemostasis with its ligand vitamin D. Consequently, we suggested that VDR might be a member of this complex also with its own non-genomic action and that it can regulate the APP processing pathway in this way in neurons.

Membrane Assembly during the Infection Cycle of the Giant Mimivirus

PubMed Central

Mutsafi, Yael; Shimoni, Eyal; Shimon, Amir; Minsky, Abraham

2013-01-01

Although extensively studied, the structure, cellular origin and assembly mechanism of internal membranes during viral infection remain unclear. By combining diverse imaging techniques, including the novel Scanning-Transmission Electron Microscopy tomography, we elucidate the structural stages of membrane biogenesis during the assembly of the giant DNA virus Mimivirus. We show that this elaborate multistage process occurs at a well-defined zone localized at the periphery of large viral factories that are generated in the host cytoplasm. Membrane biogenesis is initiated by fusion of multiple vesicles, ∼70 nm in diameter, that apparently derive from the host ER network and enable continuous supply of lipid components to the membrane-assembly zone. The resulting multivesicular bodies subsequently rupture to form large open single-layered membrane sheets from which viral membranes are generated. Membrane generation is accompanied by the assembly of icosahedral viral capsids in a process involving the hypothetical major capsid protein L425 that acts as a scaffolding protein. The assembly model proposed here reveals how multiple Mimivirus progeny can be continuously and efficiently generated and underscores the similarity between the infection cycles of Mimivirus and Vaccinia virus. Moreover, the membrane biogenesis process indicated by our findings provides new insights into the pathways that might mediate assembly of internal viral membranes in general. PMID:23737745

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

PubMed

Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

2017-04-28

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro , and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

PubMed Central

Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

2017-01-01

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo. However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis, we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro, and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. PMID:28283569

Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells

PubMed Central

1975-01-01

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane. PMID:163833

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.

Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

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

Bonekamp, Nina A.; Vormund, Kerstin; Jacob, Ralf

2010-12-10

The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. Aftermore » silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.« less

High-Resolution pH Imaging of Living Bacterial Cells To Detect Local pH Differences

PubMed Central

Morimoto, Yusuke V.; Kami-ike, Nobunori; Miyata, Tomoko; Kawamoto, Akihiro; Kato, Takayuki

2016-01-01

ABSTRACT Protons are utilized for various biological activities such as energy transduction and cell signaling. For construction of the bacterial flagellum, a type III export apparatus utilizes ATP and proton motive force to drive flagellar protein export, but the energy transduction mechanism remains unclear. Here, we have developed a high-resolution pH imaging system to measure local pH differences within living Salmonella enterica cells, especially in close proximity to the cytoplasmic membrane and the export apparatus. The local pH near the membrane was ca. 0.2 pH unit higher than the bulk cytoplasmic pH. However, the local pH near the export apparatus was ca. 0.1 pH unit lower than that near the membrane. This drop of local pH depended on the activities of both transmembrane export components and FliI ATPase. We propose that the export apparatus acts as an H+/protein antiporter to couple ATP hydrolysis with H+ flow to drive protein export. PMID:27923921

Localization of sialic acid in kidney glomeruli: regionalization in the podocyte plasma membrane and loss in experimental nephrosis.

PubMed

Charest, P M; Roth, J

1985-12-01

Sialic acid residues were localized by electron microscopy in renal glomeruli of normal and puromycin-treated rats with a cytochemical technique that utilized the Limax flavus lectin. In Lowicryl K4M thin sections from normal rats, sialic acid residues were found along the plasma membrane of the various glomerular cell types and in the glomerular basement membrane as well as the mesangial matrix. In NaDodSO4/PAGE, sialic acid residues of normal glomeruli were mainly confined to a 140-kDa protein previously identified as podocalyxin. The distribution of sialic acid residues in the podocyte plasma membrane was found to be remarkably regionalized. Based on the differential labeling intensity, three plasma membrane domains could be defined: the foot process base, the foot process region above the slit diaphragm, and the body of podocytes. Cytochemical and biochemical analysis of glomeruli from puromycin-treated rats showed a loss of sialic acid residues from glomerular sialoglycoconjugates indicating a perturbated glycosylation.

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts.

PubMed

Ring, Axel; Le Lay, Soazig; Pohl, Juergen; Verkade, Paul; Stremmel, Wolfgang

2006-04-01

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.

In silico local structure approach: a case study on outer membrane proteins.

PubMed

Martin, Juliette; de Brevern, Alexandre G; Camproux, Anne-Claude

2008-04-01

The detection of Outer Membrane Proteins (OMP) in whole genomes is an actual question, their sequence characteristics have thus been intensively studied. This class of protein displays a common beta-barrel architecture, formed by adjacent antiparallel strands. However, due to the lack of available structures, few structural studies have been made on this class of proteins. Here we propose a novel OMP local structure investigation, based on a structural alphabet approach, i.e., the decomposition of 3D structures using a library of four-residue protein fragments. The optimal decomposition of structures using hidden Markov model results in a specific structural alphabet of 20 fragments, six of them dedicated to the decomposition of beta-strands. This optimal alphabet, called SA20-OMP, is analyzed in details, in terms of local structures and transitions between fragments. It highlights a particular and strong organization of beta-strands as series of regular canonical structural fragments. The comparison with alphabets learned on globular structures indicates that the internal organization of OMP structures is more constrained than in globular structures. The analysis of OMP structures using SA20-OMP reveals some recurrent structural patterns. The preferred location of fragments in the distinct regions of the membrane is investigated. The study of pairwise specificity of fragments reveals that some contacts between structural fragments in beta-sheets are clearly favored whereas others are avoided. This contact specificity is stronger in OMP than in globular structures. Moreover, SA20-OMP also captured sequential information. This can be integrated in a scoring function for structural model ranking with very promising results. (c) 2007 Wiley-Liss, Inc.

The Role of Membrane Curvature in Nanoscale Topography-Induced Intracellular Signaling.

PubMed

Lou, Hsin-Ya; Zhao, Wenting; Zeng, Yongpeng; Cui, Bianxiao

2018-05-15

Over the past decade, there has been growing interest in developing biosensors and devices with nanoscale and vertical topography. Vertical nanostructures induce spontaneous cell engulfment, which enhances the cell-probe coupling efficiency and the sensitivity of biosensors. Although local membranes in contact with the nanostructures are found to be fully fluidic for lipid and membrane protein diffusions, cells appear to actively sense and respond to the surface topography presented by vertical nanostructures. For future development of biodevices, it is important to understand how cells interact with these nanostructures and how their presence modulates cellular function and activities. How cells recognize nanoscale surface topography has been an area of active research for two decades before the recent biosensor works. Extensive studies show that surface topographies in the range of tens to hundreds of nanometers can significantly affect cell functions, behaviors, and ultimately the cell fate. For example, titanium implants having rough surfaces are better for osteoblast attachment and host-implant integration than those with smooth surfaces. At the cellular level, nanoscale surface topography has been shown by a large number of studies to modulate cell attachment, activity, and differentiation. However, a mechanistic understanding of how cells interact and respond to nanoscale topographic features is still lacking. In this Account, we focus on some recent studies that support a new mechanism that local membrane curvature induced by nanoscale topography directly acts as a biochemical signal to induce intracellular signaling, which we refer to as the curvature hypothesis. The curvature hypothesis proposes that some intracellular proteins can recognize membrane curvatures of a certain range at the cell-to-material interface. These proteins then recruit and activate downstream components to modulate cell signaling and behavior. We discuss current technologies

Recycling of used perfluorosulfonic acid membranes

DOEpatents

Grot, Stephen [Middletown, DE; Grot, Walther [Chadds Ford, PA

2007-08-14

A method for recovering and recycling catalyst coated fuel cell membranes includes dissolving the used membranes in water and solvent, heating the dissolved membranes under pressure and separating the components. Active membranes are produced from the recycled materials.

Stimulated Emission Depletion Live-Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

PubMed Central

Wagner, Eva; Lauterbach, Marcel A.; Kohl, Tobias; Westphal, Volker; Williams, George S.B.; Steinbrecher, Julia H.; Streich, Jan-Hendrik; Korff, Brigitte; Tuan, Hoang-Trong M.; Hagen, Brian; Luther, Stefan; Hasenfuss, Gerd; Parlitz, Ulrich; Jafri, M. Saleet; Hell, Stefan W.; Lederer, W. Jonathan; Lehnart, Stephan E.

2014-01-01

Rationale Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing. PMID:22723297

Cooperative interactions of LPPR family members in membrane localization and alteration of cellular morphology

PubMed Central

Yu, Panpan; Agbaegbu, Chinyere; Malide, Daniela A.; Wu, Xufeng; Katagiri, Yasuhiro; Hammer, John A.; Geller, Herbert M.

2015-01-01

ABSTRACT The lipid phosphate phosphatase-related proteins (LPPRs), also known as plasticity-related genes (PRGs), are classified as a new brain-enriched subclass of the lipid phosphate phosphatase (LPP) superfamily. They induce membrane protrusions, neurite outgrowth or dendritic spine formation in cell lines and primary neurons. However, the exact roles of LPPRs and the mechanisms underlying their effects are not certain. Here, we present the results of a large-scale proteome analysis to determine LPPR1-interacting proteins using co-immunoprecipitation coupled to mass spectrometry. We identified putative LPPR1-binding proteins involved in various biological processes. Most interestingly, we identified the interaction of LPPR1 with its family member LPPR3, LPPR4 and LPPR5. Their interactions were characterized by co-immunoprecipitation and colocalization analysis using confocal and super-resolution microscopy. Moreover, co-expressing two LPPR members mutually elevated their protein levels, facilitated their plasma membrane localization and resulted in an increased induction of membrane protrusions as well as the phosphorylation of S6 ribosomal protein. Taken together, we revealed a new functional cooperation between LPPR family members and discovered for the first time that LPPRs likely exert their function through forming complex with its family members. PMID:26183180

Impact of ticagrelor on P2Y1 and P2Y12 localization and on cholesterol levels in platelet plasma membrane.

PubMed

Rabani, Vahideh; Montange, Damien; Meneveau, Nicolas; Davani, Siamak

2017-10-11

Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management.

Screening molecular associations with lipid membranes using natural abundance 13C cross-polarization magic-angle spinning NMR and principal component analysis.

PubMed

Middleton, David A; Hughes, Eleri; Madine, Jillian

2004-08-11

We describe an NMR approach for detecting the interactions between phospholipid membranes and proteins, peptides, or small molecules. First, 1H-13C dipolar coupling profiles are obtained from hydrated lipid samples at natural isotope abundance using cross-polarization magic-angle spinning NMR methods. Principal component analysis of dipolar coupling profiles for synthetic lipid membranes in the presence of a range of biologically active additives reveals clusters that relate to different modes of interaction of the additives with the lipid bilayer. Finally, by representing profiles from multiple samples in the form of contour plots, it is possible to reveal statistically significant changes in dipolar couplings, which reflect perturbations in the lipid molecules at the membrane surface or within the hydrophobic interior.

Global Proteomic Analysis Reveals an Exclusive Role of Thylakoid Membranes in Bioenergetics of a Model Cyanobacterium

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

Liberton, Michelle; Saha, Rajib; Jacobs, Jon M.

2016-04-07

Cyanobacteria are photosynthetic microbes with highly differentiated membrane systems. These organisms contain an outer membrane, plasma membrane, and an internal system of thylakoid membranes where the photosynthetic and respiratory machinery are found. This existence of compartmentalization and differentiation of membrane systems poses a number of challenges for cyanobacterial cells in terms of organization and distribution of proteins to the correct membrane system. Proteomics studies have long sought to identify the components of the different membrane systems, and to date about 450 different proteins have been attributed to either the plasma membrane or thylakoid membrane. Given the complexity of these membranes,more » many more proteins remain to be identified in these membrane systems, and a comprehensive catalog of plasma membrane and thylakoid membrane proteins is needed. Here we describe the identification of 635 proteins in Synechocystis sp. PCC 6803 by quantitative iTRAQ isobaric labeling; of these, 459 proteins were localized to the plasma membrane and 176 were localized to the thylakoid membrane. Surprisingly, we found over 2.5 times the number of unique proteins identified in the plasma membrane compared to the thylakoid membrane. This suggests that the protein composition of the thylakoid membrane is more homogeneous than the plasma membrane, consistent with the role of the plasma membrane in diverse cellular processes including protein trafficking and nutrient import, compared to a more specialized role for the thylakoid membrane in cellular energetics. Overall, the protein composition of the Synechocystis 6803 plasma membrane and thylakoid membrane is quite similar to the E.coli plasma membrane and Arabidopsis thylakoid membrane, respectively. Synechocystis 6803 can therefore be described as a gram-negative bacterium that has an additional internal membrane system that fulfils the energetic requirements of the cell.« less

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

PubMed Central

Yoshida, Aiko; Sakai, Nobuaki; Uekusa, Yoshitsugu; Imaoka, Yuka; Itagaki, Yoshitsuna; Suzuki, Yuki

2018-01-01

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME. PMID:29723197

Membrane association and localization dynamics of the Ebola virus matrix protein VP40.

PubMed

Gc, Jeevan B; Gerstman, Bernard S; Chapagain, Prem P

2017-10-01

The Ebola virus matrix protein VP40 is a major structural protein that provides the scaffolding for new Ebola virus particles. For this, VP40 is first trafficked to the lower leaflet of the plasma membrane (PM) in its dimeric form. Once associated with the PM, the VP40 dimers undergo structural rearrangements and oligomerize into hexamers and filaments that make up the virus matrix. Therefore, association of the VP40 dimers and their stabilization at the PM is a crucial step in the Ebola life-cycle. To understand the molecular details of the VP40 dimer-PM interactions, we investigated the dimer association with the inner leaflet of the PM using detailed all-atom molecular dynamics (MD) simulations. The formation of the dimer-PM complex is facilitated by the interactions of the VP40 lysine residues and the anionic lipids POPS, POPI, and PIP 2 in the PM. In contrast, the dimer fails to associate with a membrane without POPS, POPI, or PIP 2 lipids. We explored the mechanisms of the association and identified important residues and lipids involved in localization and stabilization of VP40 dimers at the PM. MD simulations elucidate the role of a C-terminal α-helix alignment parallel to the lipid bilayer surface as well as the creation of membrane defects that allow partial insertion of the hydrophobic residue V276 into the membrane to further stabilize the VP40 dimer-PM complex. Understanding the mechanisms of the VP40 dimer-PM association that facilitate oligomerization can be important for potentially targeting the VP40 for small molecules that can interfere with the virus life-cycle. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening antiallergic components from Carthamus tinctorius using rat basophilic leukemia 2H3 cell membrane chromatography combined with high-performance liquid chromatography and tandem mass spectrometry.

PubMed

Han, Shengli; Huang, Jing; Cui, Ronghua; Zhang, Tao

2015-02-01

Carthamus tinctorius, used in traditional Chinese medicine, has many pharmacological effects, such as anticoagulant effects, antioxidant effects, antiaging effects, regulation of gene expression, and antitumor effects. However, there is no report on the antiallergic effects of the components in C. tinctorius. In the present study, we investigated the antiallergic components of C. tinctorius and its mechanism of action. A rat basophilic leukemia 2H3/cell membrane chromatography coupled online with high-performance liquid chromatography and tandem mass spectrometry method was developed to screen antiallergic components from C. tinctorius. The screening results showed that Hydroxysafflor yellow A, from C. tinctorius, was the targeted component that retained on the rat basophilic leukemia 2H3/cell membrane chromatography column. We measured the amount of β-hexosaminidase and histamine released in mast cells and the key markers of degranulation. The release assays showed that Hydroxysafflor yellow A could attenuate the immunoglobulin E induced release of allergic cytokines without affecting cell viability from 1.0 to 50.0 μM. In conclusion, the established rat basophilic leukemia 2H3 cell membrane chromatography coupled with online high-performance liquid chromatography and tandem mass spectrometry method successfully screened and identified Hydroxysafflor yellow A from C. tinctorius as a potential antiallergic component. Pharmacological analysis elucidated that Hydroxysafflor yellow A is an effective natural component for inhibiting immunoglobulin E-antigen-mediated degranulation. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Membrane-augmented cryogenic methane/nitrogen separation

DOEpatents

Lokhandwala, Kaaeid

1997-01-01

A membrane separation process combined with a cryogenic separation process for treating a gas stream containing methane, nitrogen and at least one other component. The membrane separation process works by preferentially permeating methane and the other component and rejecting nitrogen. The process is particularly useful in removing components such as water, carbon dioxide or C.sub.3+ hydrocarbons that might otherwise freeze and plug the cryogenic equipment.

The C-terminus of the oncoprotein TGAT is necessary for plasma membrane association and efficient RhoA-mediated signaling.

PubMed

van Unen, J; Botman, D; Yin, T; Wu, Y I; Hink, M A; Gadella, T W J; Postma, M; Goedhart, J

2018-06-07

Rho guanine exchange factors (RhoGEFs) control cellular processes such as migration, adhesion and proliferation. Alternative splicing of the RhoGEF Trio produces TGAT. The RhoGEF TGAT is an oncoprotein with constitutive RhoGEF activity. We investigated whether the subcellular location of TGAT is critical for its RhoGEF activity. Since plasma membrane associated RhoGEFs are particularly effective at activating RhoA, plasma membrane localization of TGAT was examined. To this end, we developed a highly sensitive image analysis method to quantitatively measure plasma membrane association. The method requires a cytoplasmic marker and a plasma membrane marker, which are co-imaged with the tagged protein of interest. Linear unmixing is performed to determine the plasma membrane and cytoplasmic component in the fluorescence signal of protein of interest. The analysis revealed that wild-type TGAT is partially co-localized with the plasma membrane. Strikingly, cysteine TGAT-mutants lacking one or more putative palmitoylation sites in the C-tail, still showed membrane association. In contrast, a truncated variant, lacking the last 15 amino acids, TGAT Δ15 , lost membrane association. We show that membrane localization of TGAT was responsible for high RhoGEF activity by using a RhoA FRET-sensor and by determining F-actin levels. Mutants of TGAT that still maintained membrane association showed similar activity as wild-type TGAT. In contrast, the activity was abrogated for the cytoplasmic TGAT Δ15 variant. Synthetic recruitment of TGAT Δ15 to membranes confirmed that TGAT effectively activates RhoA at the plasma membrane. Together, these results show that membrane association of TGAT is critical for its activity.

ABC-transporters are localized in caveolin-1-positive and reggie-1-negative and reggie-2-negative microdomains of the canalicular membrane in rat hepatocytes.

PubMed

Ismair, Manfred G; Häusler, Stephanie; Stuermer, Claudia A; Guyot, Christelle; Meier, Peter J; Roth, Jürgen; Stieger, Bruno

2009-05-01

The canalicular plasma membrane is constantly exposed to bile acids acting as detergents. Bile acids are essential to mediate release of biliary lipids from the canalicular membrane. Membrane microdomains (previously called lipid rafts) are biochemically defined by their resistance to detergent solubilization at cold temperature. We aimed to investigate the canalicular plasma membrane for the presence of microdomains, which could protect this membrane against the detergent action of bile acids. Highly purified rat liver canalicular plasma membrane vesicles were extracted with 1% Triton X-100 or 1% Lubrol WX at 4 degrees C and subjected to flotation through sucrose step gradients. Both detergents yielded detergent-resistant membranes containing the microdomain markers alkaline phosphatase and sphingomyelin. However, cholesterol was resistant to Lubrol WX solubilization, whereas it was only marginally resistant to solubilization by Triton X-100. The microdomain marker caveolin-1 was localized to the canalicular plasma membrane domain and was resistant to Lubrol WX, but to a large extent solubilized by Triton X-100. The two additional microdomain markers, reggie-1 and reggie-2, were localized to the basolateral and canalicular plasma membrane and were partially resistant to Lubrol WX but resistant to Triton X-100. The canalicular transporters bile salt export pump, multidrug resistance protein 2, multidrug resistance-associated protein 2, and Abcg5 were largely resistant to Lubrol WX but were solubilized by Triton X-100. These results indicate the presence of two different types of microdomains in the canalicular plasma membrane: "Lubrol-microdomains" and "Triton-microdomains". "Lubrol-microdomains" contain the machinery for canalicular bile formation and may be the starting place for canalicular lipid secretion.

EBS7 is a plant-specific component of a highly conserved endoplasmic reticulum-associated degradation system in Arabidopsis

PubMed Central

Liu, Yidan; Zhang, Congcong; Wang, Dinghe; Su, Wei; Liu, Linchuan; Wang, Muyang; Li, Jianming

2015-01-01

Endoplasmic reticulum (ER)-associated degradation (ERAD) is an essential part of an ER-localized protein quality-control system for eliminating terminally misfolded proteins. Recent studies have demonstrated that the ERAD machinery is conserved among yeast, animals, and plants; however, it remains unknown if the plant ERAD system involves plant-specific components. Here we report that the Arabidopsis ethyl methanesulfonate-mutagenized brassinosteroid-insensitive 1 suppressor 7 (EBS7) gene encodes an ER membrane-localized ERAD component that is highly conserved in land plants. Loss-of-function ebs7 mutations prevent ERAD of brassinosteroid insensitive 1-9 (bri1-9) and bri1-5, two ER-retained mutant variants of the cell-surface receptor for brassinosteroids (BRs). As a result, the two mutant receptors accumulate in the ER and consequently leak to the plasma membrane, resulting in the restoration of BR sensitivity and phenotypic suppression of the bri1-9 and bri1-5 mutants. EBS7 accumulates under ER stress, and its mutations lead to hypersensitivity to ER and salt stresses. EBS7 interacts with the ER membrane-anchored ubiquitin ligase Arabidopsis thaliana HMG-CoA reductase degradation 1a (AtHrd1a), one of the central components of the Arabidopsis ERAD machinery, and an ebs7 mutation destabilizes AtHrd1a to reduce polyubiquitination of bri1-9. Taken together, our results uncover a plant-specific component of a plant ERAD pathway and also suggest its likely biochemical function. PMID:26371323

Localized Principal Component Analysis based Curve Evolution: A Divide and Conquer Approach

PubMed Central

Appia, Vikram; Ganapathy, Balaji; Yezzi, Anthony; Faber, Tracy

2014-01-01

We propose a novel localized principal component analysis (PCA) based curve evolution approach which evolves the segmenting curve semi-locally within various target regions (divisions) in an image and then combines these locally accurate segmentation curves to obtain a global segmentation. The training data for our approach consists of training shapes and associated auxiliary (target) masks. The masks indicate the various regions of the shape exhibiting highly correlated variations locally which may be rather independent of the variations in the distant parts of the global shape. Thus, in a sense, we are clustering the variations exhibited in the training data set. We then use a parametric model to implicitly represent each localized segmentation curve as a combination of the local shape priors obtained by representing the training shapes and the masks as a collection of signed distance functions. We also propose a parametric model to combine the locally evolved segmentation curves into a single hybrid (global) segmentation. Finally, we combine the evolution of these semilocal and global parameters to minimize an objective energy function. The resulting algorithm thus provides a globally accurate solution, which retains the local variations in shape. We present some results to illustrate how our approach performs better than the traditional approach with fully global PCA. PMID:25520901

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

Perkinsus marinus superoxide dismutase 2 (PmSOD2) localizes to single-membrane subcellular compartments

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

Fernandez-Robledo, Jose A.; Schott, Eric J.; Vasta, Gerardo R.

2008-10-17

Perkinsus marinus (Phylum Perkinsozoa), a protozoan parasite of oysters, is considered one of the earliest diverging groups of the lineage leading to dinoflagellates. Perkinsus trophozoites are phagocytosed by oyster hemocytes, where they are likely exposed to reactive oxygen species. As part of its reactive oxygen detoxifying pathway, P. marinus possesses two iron-cofactored SOD (PmSOD1 and PmSOD2). Immunoflourescence analysis of P. marinus trophozoites and gene complementation in yeast revealed that PmSOD1 is targeted to the mitochondria. Surprisingly, although PmSOD2 is characterized by a bipartite N-terminus extension typical of plastid targeting, in preliminary immunofluorescence studies it was visualized as punctuate regions inmore » the cytoplasm that could not be assigned to any organelle. Here, we used immunogold electron microscopy to examine the subcellular localization PmSOD2 in P. marinus trophozoites. Gold grains were mostly associated with single-membrane vesicle-like structures, and eventually, localized to electron-dense, apparently amorphous material present in the lumen of a larger, unique compartment. The images suggested that PmSOD2 is targeted to small vesicles that fuse and/or discharge their content into a larger compartment, possibly the large vacuole typical of the mature trophozoites. In light of the in silico targeting prediction, the association of PmSOD2 with single-membrane compartments raises interesting questions regarding its organellar targeting, and the nature of a putative relic plastid in Perkinsus species.« less

Membrane Contact Sites: Complex Zones for Membrane Association and Lipid Exchange

PubMed Central

Quon, Evan; Beh, Christopher T.

2015-01-01

Lipid transport between membranes within cells involves vesicle and protein carriers, but as agents of nonvesicular lipid transfer, the role of membrane contact sites has received increasing attention. As zones for lipid metabolism and exchange, various membrane contact sites mediate direct associations between different organelles. In particular, membrane contact sites linking the plasma membrane (PM) and the endoplasmic reticulum (ER) represent important regulators of lipid and ion transfer. In yeast, cortical ER is stapled to the PM through membrane-tethering proteins, which establish a direct connection between the membranes. In this review, we consider passive and facilitated models for lipid transfer at PM–ER contact sites. Besides the tethering proteins, we examine the roles of an additional repertoire of lipid and protein regulators that prime and propagate PM–ER membrane association. We conclude that instead of being simple mediators of membrane association, regulatory components of membrane contact sites have complex and multilayered functions. PMID:26949334

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

Annexins in plasma membrane repair.

PubMed

Boye, Theresa Louise; Nylandsted, Jesper

2016-10-01

Disruption of the plasma membrane poses deadly threat to eukaryotic cells and survival requires a rapid membrane repair system. Recent evidence reveal various plasma membrane repair mechanisms, which are required for cells to cope with membrane lesions including membrane fusion and replacement strategies, remodeling of cortical actin cytoskeleton and vesicle wound patching. Members of the annexin protein family, which are Ca2+-triggered phospholipid-binding proteins emerge as important components of the plasma membrane repair system. Here, we discuss the mechanisms of plasma membrane repair involving annexins spanning from yeast to human cancer cells.

Sensing Size through Clustering in Non-Equilibrium Membranes and the Control of Membrane-Bound Enzymatic Reactions

PubMed Central

Vagne, Quentin; Turner, Matthew S.; Sens, Pierre

2015-01-01

The formation of dynamical clusters of proteins is ubiquitous in cellular membranes and is in part regulated by the recycling of membrane components. We show, using stochastic simulations and analytic modeling, that the out-of-equilibrium cluster size distribution of membrane components undergoing continuous recycling is strongly influenced by lateral confinement. This result has significant implications for the clustering of plasma membrane proteins whose mobility is hindered by cytoskeletal “corrals” and for protein clustering in cellular organelles of limited size that generically support material fluxes. We show how the confinement size can be sensed through its effect on the size distribution of clusters of membrane heterogeneities and propose that this could be regulated to control the efficiency of membrane-bound reactions. To illustrate this, we study a chain of enzymatic reactions sensitive to membrane protein clustering. The reaction efficiency is found to be a non-monotonic function of the system size, and can be optimal for sizes comparable to those of cellular organelles. PMID:26656912

Steerable Principal Components for Space-Frequency Localized Images*

PubMed Central

Landa, Boris; Shkolnisky, Yoel

2017-01-01

As modern scientific image datasets typically consist of a large number of images of high resolution, devising methods for their accurate and efficient processing is a central research task. In this paper, we consider the problem of obtaining the steerable principal components of a dataset, a procedure termed “steerable PCA” (steerable principal component analysis). The output of the procedure is the set of orthonormal basis functions which best approximate the images in the dataset and all of their planar rotations. To derive such basis functions, we first expand the images in an appropriate basis, for which the steerable PCA reduces to the eigen-decomposition of a block-diagonal matrix. If we assume that the images are well localized in space and frequency, then such an appropriate basis is the prolate spheroidal wave functions (PSWFs). We derive a fast method for computing the PSWFs expansion coefficients from the images' equally spaced samples, via a specialized quadrature integration scheme, and show that the number of required quadrature nodes is similar to the number of pixels in each image. We then establish that our PSWF-based steerable PCA is both faster and more accurate then existing methods, and more importantly, provides us with rigorous error bounds on the entire procedure. PMID:29081879

Secretins revealed: structural insights into the giant gated outer membrane portals of bacteria.

PubMed

Majewski, Dorothy D; Worrall, Liam J; Strynadka, Natalie Cj

2018-03-23

The acquisition and evolution of customized and often highly complex secretion systems allows Gram-negative bacteria to efficiently passage large macromolecules across both inner and outer membranes and, in some cases, that of the infected host. Essential to the virulence and ultimate survival of the many pathogenic species that encode them, secretion systems export a wide variety of effector proteins and DNA as well as the downstream extracellular filaments of the secretion apparatus themselves. Although these customized secretion systems differ in their cytosolic and inner membrane components, several commonly rely on the secretin family of giant pores to allow these large substrates to traverse the outer membrane. Recently, several near-atomic resolution cryo-EM secretin structures have unveiled the first insights into the unique structural motifs required for outer membrane localization, assembly, hallmark ultrastable nature, spontaneous membrane insertion, and mechanism of action-including the requisite central gating needed to prevent deleterious passage of periplasmic contents to the extracellular space. Copyright © 2018. Published by Elsevier Ltd.

Membrane Localization of β-Amyloid 1–42 in Lysosomes

PubMed Central

Liu, Rui-Qin; Zhou, Qing-Hua; Ji, Shang-Rong; Zhou, Qiang; Feng, Du; Wu, Yi; Sui, Sen-Fang

2010-01-01

β-Amyloid peptide (Aβ42) is the core protein of amyloid plaque in Alzheimer disease. The intracellular accumulation of Aβ42 in the endosomal/lysosomal system has been under investigation for many years, but the direct link between Aβ42 accumulation and dysfunction of the endosomal/lysosomal system is still largely unknown. Here, we found that both in vitro and in vivo, a major portion of Aβ42 was tightly inserted into and a small portion peripherally associated with the lysosomal membrane, whereas its soluble portion was minimal. We also found that the Aβ42 molecules inserted into the membrane tended to form multiple oligomeric aggregates, whereas Aβ40 peptides formed only dimers. Neutralizing lysosomal pH in differentiated PC12 cells decreased the lysosomal membrane insertion of Aβ42 and moderated Aβ42-induced lysosomal labilization and cytotoxicity. Our findings, thus, suggest that the membrane-inserted portion of Aβ42 accumulated in lysosomes may destabilize the lysosomal membrane and induce neurotoxicity. PMID:20430896

Experimental dissection of oxygen transport resistance in the components of a polymer electrolyte membrane fuel cell

NASA Astrophysics Data System (ADS)

Oh, Hwanyeong; Lee, Yoo il; Lee, Guesang; Min, Kyoungdoug; Yi, Jung S.

2017-03-01

Oxygen transport resistance is a major obstacle for obtaining high performance in a polymer electrolyte membrane fuel cell (PEMFC). To distinguish the major components that inhibit oxygen transport, an experimental method is established to dissect the oxygen transport resistance of the components of the PEMFC, such as the substrate, micro-porous layer (MPL), catalyst layer, and ionomer film. The Knudsen numbers are calculated to determine the types of diffusion mechanisms at each layer by measuring the pore sizes with either mercury porosimetry or BET analysis. At the under-saturated condition where condensation is mostly absent, the molecular diffusion resistance is dissected by changing the type of inert gas, and ionomer film permeation is separated by varying the inlet gas humidity. Moreover, the presence of the MPL and the variability of the substrate thickness allow the oxygen transport resistance at each component of a PEMFC to be dissected. At a low relative humidity of 50% and lower, an ionomer film had the largest resistance, while the contribution of the MPL was largest for the other humidification conditions.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 2. QSAR models that predict localization of fluorescent probes used to identify ("specifically stain") various biomembranes and membranous organelles.

PubMed

Horobin, R W; Stockert, J C; Rashid-Doubell, F

2015-05-01

We discuss a variety of biological targets including generic biomembranes and the membranes of the endoplasmic reticulum, endosomes/lysosomes, Golgi body, mitochondria (outer and inner membranes) and the plasma membrane of usual fluidity. For each target, we discuss the access of probes to the target membrane, probe uptake into the membrane and the mechanism of selectivity of the probe uptake. A statement of the QSAR decision rule that describes the required physicochemical features of probes that enable selective staining also is provided, followed by comments on exceptions and limits. Examples of probes typically used to demonstrate each target structure are noted and decision rule tabulations are provided for probes that localize in particular targets; these tabulations show distribution of probes in the conceptual space defined by the relevant structure parameters ("parameter space"). Some general implications and limitations of the QSAR models for probe targeting are discussed including the roles of certain cell and protocol factors that play significant roles in lipid staining. A case example illustrates the predictive ability of QSAR models. Key limiting values of the head group hydrophilicity parameter associated with membrane-probe interactions are discussed in an appendix.

Plasmodium falciparum FIKK Kinase Members Target Distinct Components of the Erythrocyte Membrane

PubMed Central

Scheidig-Benatar, Christine; Cooke, Brian M.; Scherf, Artur

2010-01-01

Background Modulation of infected host cells by intracellular pathogens is a prerequisite for successful establishment of infection. In the human malaria parasite Plasmodium falciparum, potential candidates for erythrocyte remodelling include the apicomplexan-specific FIKK kinase family (20 members), several of which have been demonstrated to be transported into the erythrocyte cytoplasm via Maurer's clefts. Methodology In the current work, we have knocked out two members of this gene family (Pf fikk7.1 and Pf fikk12), whose products are localized at the inner face of the erythrocyte membrane. Both mutant parasite lines were viable and erythrocytes infected with these parasites showed no detectable alteration in their ability to adhere in vitro to endothelial receptors such as chondroitin sulfate A and CD36. However, we observed sizeable decreases in the rigidity of infected erythrocytes in both knockout lines. Mutant parasites were further analyzed using a phospho-proteomic approach, which revealed distinct phosphorylation profiles in ghost preparations of infected erythrocytes. Knockout parasites showed a significant reduction in the level of phosphorylation of a protein of approximately 80 kDa for FIKK12-KO in trophozoite stage and a large protein of about 300 kDa for FIKK7.1-KO in schizont stage. Conclusions Our results suggest that FIKK members phosphorylate different membrane skeleton proteins of the infected erythrocyte in a stage-specific manner, inducing alterations in the mechanical properties of the parasite-infected red blood cell. This suggests that these host cell modifications may contribute to the parasites' survival in the circulation of the human host. PMID:20668526

Plasmodium falciparum FIKK kinase members target distinct components of the erythrocyte membrane.

PubMed

Nunes, Marta C; Okada, Mami; Scheidig-Benatar, Christine; Cooke, Brian M; Scherf, Artur

2010-07-23

Modulation of infected host cells by intracellular pathogens is a prerequisite for successful establishment of infection. In the human malaria parasite Plasmodium falciparum, potential candidates for erythrocyte remodelling include the apicomplexan-specific FIKK kinase family (20 members), several of which have been demonstrated to be transported into the erythrocyte cytoplasm via Maurer's clefts. In the current work, we have knocked out two members of this gene family (Pf fikk7.1 and Pf fikk12), whose products are localized at the inner face of the erythrocyte membrane. Both mutant parasite lines were viable and erythrocytes infected with these parasites showed no detectable alteration in their ability to adhere in vitro to endothelial receptors such as chondroitin sulfate A and CD36. However, we observed sizeable decreases in the rigidity of infected erythrocytes in both knockout lines. Mutant parasites were further analyzed using a phospho-proteomic approach, which revealed distinct phosphorylation profiles in ghost preparations of infected erythrocytes. Knockout parasites showed a significant reduction in the level of phosphorylation of a protein of approximately 80 kDa for FIKK12-KO in trophozoite stage and a large protein of about 300 kDa for FIKK7.1-KO in schizont stage. Our results suggest that FIKK members phosphorylate different membrane skeleton proteins of the infected erythrocyte in a stage-specific manner, inducing alterations in the mechanical properties of the parasite-infected red blood cell. This suggests that these host cell modifications may contribute to the parasites' survival in the circulation of the human host.

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

PubMed Central

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

2013-01-01

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

Diffusion of molecules and macromolecules in thylakoid membranes.

PubMed

Kirchhoff, Helmut

2014-04-01

The survival and fitness of photosynthetic organisms is critically dependent on the flexible response of the photosynthetic machinery, harbored in thylakoid membranes, to environmental changes. A central element of this flexibility is the lateral diffusion of membrane components along the membrane plane. As demonstrated, almost all functions of photosynthetic energy conversion are dependent on lateral diffusion. The mobility of both small molecules (plastoquinone, xanthophylls) as well as large protein supercomplexes is very sensitive to changes in structural boundary conditions. Knowledge about the design principles that govern the mobility of photosynthetic membrane components is essential to understand the dynamic response of the photosynthetic machinery. This review summarizes our knowledge about the factors that control diffusion in thylakoid membranes and bridges structural membrane alterations to changes in mobility and function. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components. Copyright © 2013 Elsevier B.V. All rights reserved.

Shape matters in protein mobility within membranes

PubMed Central

Quemeneur, François; Sigurdsson, Jon K.; Renner, Marianne; Atzberger, Paul J.; Bassereau, Patricia; Lacoste, David

2014-01-01

The lateral mobility of proteins within cell membranes is usually thought to be dependent on their size and modulated by local heterogeneities of the membrane. Experiments using single-particle tracking on reconstituted membranes demonstrate that protein diffusion is significantly influenced by the interplay of membrane curvature, membrane tension, and protein shape. We find that the curvature-coupled voltage-gated potassium channel (KvAP) undergoes a significant increase in protein mobility under tension, whereas the mobility of the curvature-neutral water channel aquaporin 0 (AQP0) is insensitive to it. Such observations are well explained in terms of an effective friction coefficient of the protein induced by the local membrane deformation. PMID:24706877

Membrane-augmented cryogenic methane/nitrogen separation

DOEpatents

Lokhandwala, K.

1997-07-15

A membrane separation process is described which is combined with a cryogenic separation process for treating a gas stream containing methane, nitrogen and at least one other component. The membrane separation process works by preferentially permeating methane and the other component and rejecting nitrogen. The process is particularly useful in removing components such as water, carbon dioxide or C{sub +2} hydrocarbons that might otherwise freeze and plug the cryogenic equipment. 10 figs.

Implementation of CUAHSI-HIS Community Project Components in a Local Observatory

NASA Astrophysics Data System (ADS)

Muste, M.; Arnold, N.; Kim, D.

2008-12-01

The deployment of the eleven WATERS Network local observatories using CUAHSI-HIS project products showed that water observations data collected by academic investigators could be stored, published on the Internet, federated with water observations data published by water agencies, and searched using a concept framework that connects with variables in each individual data source. For many within the water resources community, the CUAHSI-HIS community project represents a new opportunity to approach the management, publication, and analysis of their data systematically - i.e., moving from collections of ASCII text or spreadsheet files to relational data models. This research describes the initial efforts carried out by a University of Iowa research group during the component implementation of a hydrologic community project in a local CI-based digital watershed (DW). The goal was to test what types of data query the DW can handle and see how it performs in use cases where data streams are coupled with models for continuous forecasting. This paper also discusses the general context for the DW development and summarizes the lessons learned by the group during this initial developmental stage. Given the uniform and scalable nature of the community project components, it is expected that the workflows presented herein are transferable to other users and other watersheds.

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

Altered Subcellular Localization of a Tobacco Membrane Raft-Associated Remorin Protein by Tobamovirus Infection and Transient Expression of Viral Replication and Movement Proteins

PubMed Central

Sasaki, Nobumitsu; Takashima, Eita; Nyunoya, Hiroshi

2018-01-01

Remorins are plant specific proteins found in plasma membrane microdomains (termed lipid or membrane rafts) and plasmodesmata. A potato remorin is reported to be involved in negatively regulating potexvirus movement and plasmodesmal permeability. In this study, we isolated cDNAs of tobacco remorins (NtREMs) and examined roles of an NtREM in infection by tomato mosaic virus (ToMV). Subcellular localization analysis using fluorescently tagged NtREM, ToMV, and viral replication and movement proteins (MPs) indicated that virus infection and transient expression of the viral proteins promoted the formation of NtREM aggregates by altering the subcellular distribution of NtREM, which was localized uniformly on the plasma membrane under normal conditions. NtREM aggregates were often observed associated closely with endoplasmic reticulum networks and bodies of the 126K replication and MPs. The bimolecular fluorescence complementation assay indicated that NtREM might interact directly with the MP on the plasma membrane and around plasmodesmata. In addition, transient overexpression of NtREM facilitated ToMV cell-to-cell movement. Based on these results, we discuss possible roles of the tobacco remorin in tobamovirus movement. PMID:29868075

Watching individual molecules flex within lipid membranes using SERS

NASA Astrophysics Data System (ADS)

Taylor, Richard W.; Benz, Felix; Sigle, Daniel O.; Bowman, Richard W.; Bao, Peng; Roth, Johannes S.; Heath, George R.; Evans, Stephen D.; Baumberg, Jeremy J.

2014-08-01

Interrogating individual molecules within bio-membranes is key to deepening our understanding of biological processes essential for life. Using Raman spectroscopy to map molecular vibrations is ideal to non-destructively `fingerprint' biomolecules for dynamic information on their molecular structure, composition and conformation. Such tag-free tracking of molecules within lipid bio-membranes can directly connect structure and function. In this paper, stable co-assembly with gold nano-components in a `nanoparticle-on-mirror' geometry strongly enhances the local optical field and reduces the volume probed to a few nm3, enabling repeated measurements for many tens of minutes on the same molecules. The intense gap plasmons are assembled around model bio-membranes providing molecular identification of the diffusing lipids. Our experiments clearly evidence measurement of individual lipids flexing through telltale rapid correlated vibrational shifts and intensity fluctuations in the Raman spectrum. These track molecules that undergo bending and conformational changes within the probe volume, through their interactions with the environment. This technique allows for in situ high-speed single-molecule investigations of the molecules embedded within lipid bio-membranes. It thus offers a new way to investigate the hidden dynamics of cell membranes important to a myriad of life processes.

Distributed microscopic actuation analysis of deformable plate membrane mirrors

NASA Astrophysics Data System (ADS)

Lu, Yifan; Yue, Honghao; Deng, Zongquan; Tzou, Hornsen

2018-02-01

To further reduce the areal density of optical mirrors used in space telescopes and other space-borne optical structures, the concept of flexible membrane deformable mirror has been proposed. Because of their high flexibility, poor stiffness and low damping properties, environmental excitations such as orbital maneuver, path changing, and non-uniform heating may induce unexpected vibrations and thus reduce working performance. Therefore, active vibration control is essential for these membrane mirrors. In this paper, two different mirror models, i.e., the plate membrane model and pure membrane model, are studied respectively. In order to investigate the modal vibration characteristics of the mirror, a piezoelectric layer is fully laminated on its non-reflective side to serve as actuators. Dynamic equations of the mirror laminated with piezoelectric actuators are presented first. Then, the actuator induced modal control force is defined. When the actuator area shrinks to infinitesimal, the expressions of microscopic local modal control force and its two components are obtained to predict the spatial microscopic actuation behavior of the mirror. Different membrane pretension forces are also applied to reveal the tension effects on the actuation of the mirror. Analyses indicate that the spatial distribution of modal micro-control forces is exactly the same with the sensing signals distribution of the mirror, which provides crucial guidelines for optimal actuator placement of membrane deformable mirrors.

Distributed microscopic actuation analysis of paraboloidal membrane shells of different geometric parameters

NASA Astrophysics Data System (ADS)

Yue, Honghao; Lu, Yifan; Deng, Zongquan; Tzou, Hornsen

2018-03-01

Paraboloidal membrane shells of revolution are commonly used as key components for advanced aerospace structures and aviation mechanical systems. Due to their high flexibility and low damping property, active vibration control is of significant importance for these in-orbit membrane structures. To explore the dynamic control behavior of space flexible paraboloidal membrane shells, precision distributed actuation and control effectiveness of free-floating paraboloidal membrane shells with piezoelectric actuators are investigated. Governing equations of the shell structronic system are presented first. Then, distributed control forces and control actions are formulated. A transverse mode shape function of the paraboloidal shell based on the membrane approximation theory and specified boundary condition is assumed in the modal control force analysis. The actuator induced modal control forces on the paraboloidal shell are derived. The expressions of microscopic local modal control forces are obtained by shrinking the actuator area into infinitesimal and the four control components are investigated respectively to predict the spatial microscopic actuation behavior. Geometric parameter (height-radius ratio and shell thickness) effects on the modal actuation behavior are explored when evaluating the micro-control efficiency. Four different cases are discussed and the results reveal the fact that shallow (e.g., antennas/reflectors) and deep (e.g., rocket/missile fairing) paraboloidal shells exhibit totally different modal actuation behaviors due to their curvature differences. Analytical results in this paper can serve as guidelines for optimal actuator placement for vibration control of different paraboloidal structures.

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.

Characterization of pneumolysin from Streptococcus pneumoniae, interacting with carbohydrate moiety and cholesterol as a component of cell membrane.

PubMed

Lim, Jong Eun; Park, Seong Ah; Bong, Seoung Min; Chi, Young Min; Lee, Ki Seog

2013-01-11

The cytolytic mechanism of cholesterol-dependent cytolysins (CDCs) requires the presence of cholesterol in the target cell membrane. Membrane cholesterol was thought to serve as the common receptor for these toxins, but not all CDCs require cholesterol for binding. One member of this toxin family, pneumolysin (PLY) is a major virulence factor of Streptococcus pneumoniae, and the mechanism via which PLY binds to its putative receptor or cholesterol on the cell membrane is still poorly understood. Here, we demonstrated that PLY interacted with carbohydrate moiety and cholesterol as a component of the cell membrane, using the inhibitory effect of hemolytic activity. The hemolytic activity of PLY was inhibited by cholesterol-MβCD, which is in a 3β configuration at the C3-hydroxy group, but is not in a 3α-configuration. In the interaction between PLY and carbohydrate moiety, the mannose showed a dose-dependent increase in the inhibition of PLY hemolytic activity. The binding ability of mannose with truncated PLYs, as determined by the pull-down assay, showed that mannose might favor binding to domain 4 rather than domains 1-3. These studies provide a new model for the mechanism of cellular recognition by PLY, as well as a foundation for future investigations into whether non-sterol molecules can serve as receptors for other members of the CDC family of toxins. Copyright © 2012 Elsevier Inc. All rights reserved.

Polar localization of plasma membrane Ca2+/Mg2+ ATPase correlates with the pattern of steady ionic currents in eggs ofLymnaea stagnalis andBithynia tentaculata (Mollusca).

PubMed

Zivkovic, Danica; Créton, Robbert; Zwaan, Gideon; de Bruijn, Willem C; Dohmen, M René

1990-11-01

During extrusion of the first polar body in eggs ofLymnaea stagnalis andBithynia tentaculata a localized Ca 2+ /Mg 2+ ATPase activity was detected, using Ando's enzyme-cytochemical method for electron microscopy [Ando et al. (1981) Acta Histochem Cytochem 14:705-726]. The enzyme activity was distributed in a polar fashion, along the cytoplasmic face of the plasma membrane. In the eggs ofLymnaea it was found only in the vegetal hemisphere, whereas inBithynia eggs it was localized both in the vegetal hemisphere and at the animal pole. This pattern of enzyme activity corresponds to the polar pattern of transcellular ionic currents measured with the vibrating probe, which we showed to be partially carried or regulated by calcium [Zivkovic and Dohmen (1989) Biol Bull (Woods Hole) 176 (Suppl):103-109]. The characteristics of the ATPase were studied using a variety of approaches such as ion and substrate depletions and substitutions, addition of specific inhibitors of ATPase activity, treatment with EDTA/EGTA and electron energy-loss spectrometry. The results indicate that, inLymnaea, there are at least two enzymatic entities. The first one is a Ca 2+ /Mg 2+ ATPase localized along the membrane and in the cortex of the vegetal hemisphere. The second one is a Ca 2+ -stimulated ATPase (calcium pump of the plasma membrane) localized in a small region of the membrane at the vegetal pole. We speculate that in the eggs ofLymnaea andBithynia a functional relationship exists between the plasma-membrane-associated ATPase activity and the transcellular ionic currents measured in the same region.

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

PubMed

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

2018-06-27

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

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.

Radiation effects on bovine taste bud membranes

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

Shatzman, A.R.; Mossman, K.L.

1982-11-01

In order to investigate the mechanisms of radiation-induced taste loss, the effects of radiation on preparations of enriched bovine taste bud membranes were studied. Taste buds containing circumvallate papilae, and surrounding control epithelial tissues devoid of taste buds, were obtained from steers and given radiation doses of 0-7000 cGy (rad). Tissue fractions were isolated into membrane-enriched and heterogeneous components using differential and sucrose gradient centrifugation of tissue homogenates. The yield of membranes, as measured by protein content in the buoyant membrane-enriched fractions, was reduced in quantity with increasing radiation dose. The relation between radiation dose and membrane quantity in membrane-enrichedmore » fractions could be fit by a simple exponential model with taste bud-derived membranes twice as radiosensitive as membranes from control epithelial tissue. Binding of sucrose, sodium, and acetate and fluoride stimulation of adenylate cyclase were nearly identical in both irradiated and nonirradiated intact membranes. Radiation had no effect on fractions of heterogeneous components. While it is not clear what changes are occurring in enriched taste cell membranes, damage to membranes may play an important role in the taste loss observed in patients following radiotherapy.« less

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

PubMed

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

2018-04-19

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

The Investigation and Development of Low Cost Hardware Components for Proton-Exchange Membrane Fuel Cells - Final Report

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

George A. Marchetti

1999-12-15

Proton exchange membrane (PEM) fuel cell components, which would have a low-cost structure in mass production, were fabricated and tested. A fuel cell electrode structure, comprising a thin layer of graphite (50 microns) and a front-loaded platinum catalyst layer (600 angstroms), was shown to produce significant power densities. In addition, a PEM bipolar plate, comprising flexible graphite, carbon cloth flow-fields and an integrated polymer gasket, was fabricated. Power densities of a two-cell unit using this inexpensive bipolar plate architecture were shown to be comparable to state-of-the-art bipolar plates.

Data-Based Locally Directed Evaluation of Vocational Education Programs. Component 5. Analysis of Community Resources Utilization.

ERIC Educational Resources Information Center

Florida State Univ., Tallahassee. Program of Vocational Education.

Part of a system by which local education agency (LEA) personnel may evaluate secondary and postsecondary vocational education programs, this fifth of eight components focuses on an analysis of the utilization of community resources. Utilization of the component is designed to open communication channels among all segments of the community so that…

Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains.

PubMed

Bücherl, Christoph A; Jarsch, Iris K; Schudoma, Christian; Segonzac, Cécile; Mbengue, Malick; Robatzek, Silke; MacLean, Daniel; Ott, Thomas; Zipfel, Cyril

2017-03-06

Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.

Confined semiflexible polymers suppress fluctuations of soft membrane tubes.

PubMed

Mirzaeifard, Sina; Abel, Steven M

2016-02-14

We use Monte Carlo computer simulations to investigate tubular membrane structures with and without semiflexible polymers confined inside. At small values of membrane bending rigidity, empty fluid and non-fluid membrane tubes exhibit markedly different behavior, with fluid membranes adopting irregular, highly fluctuating shapes and non-fluid membranes maintaining extended tube-like structures. Fluid membranes, unlike non-fluid membranes, exhibit a local maximum in specific heat as their bending rigidity increases. The peak is coincident with a transition to extended tube-like structures. We further find that confining a semiflexible polymer within a fluid membrane tube reduces the specific heat of the membrane, which is a consequence of suppressed membrane shape fluctuations. Polymers with a sufficiently large persistence length can significantly deform the membrane tube, with long polymers leading to localized bulges in the membrane that accommodate regions in which the polymer forms loops. Analytical calculations of the energies of idealized polymer-membrane configurations provide additional insight into the formation of polymer-induced membrane deformations.

Mechanical membrane for the separation of a paramagnetic constituent from a fluid

DOEpatents

Maurice, David

2017-05-02

The disclosure provides an apparatus and method for the separation of a paramagnetic component from a mixture using a mechanical membrane apparatus. The mechanical membrane comprises a supporting material having a plurality of pores where each pore is surrounded by a plurality of magnetic regions. The magnetic regions augment a magnetic field on one side of the supporting material while mitigating the field to near zero on the opposite side. In operation, a flow of fluid such as air comprising a paramagnetic component such as O.sub.2 is directed toward the mechanical membrane, and the paramagnetic component is typically attracted toward a magnetic field surrounding a pore while dimagnetic components such as N.sub.2 are generally repelled. As some portion of the fluid passes through the plurality of magnetic apertures to the opposite side of the mechanical membrane, the mechanical membrane generates a fluid enriched in the paramagnetic component. Alternately, the magnetic field may act to repel the paramagnetic component while diamagnetic components such as N.sub.2 are generally unaffected and pass to the opposite side of the mechanical membrane.

Long-range correlation of the membrane potential in neocortical neurons during slow oscillation

PubMed Central

Volgushev, Maxim; Chauvette, Sylvain; Timofeev, Igor

2012-01-01

Large amplitude slow waves are characteristic for the summary brain activity, recorded as electroencephalogram (EEG) or local field potentials (LFP), during deep stages of sleep and some types of anesthesia. Slow rhythm of the synchronized EEG reflects an alternation of active (depolarized, UP) and silent (hyperpolarized, DOWN) states of neocortical neurons. In neurons, involvement in the generalized slow oscillation results in a long-range synchronization of changes of their membrane potential as well as their firing. Here, we aimed at intracellular analysis of details of this synchronization. We asked which components of neuronal activity exhibit long-range correlations during the synchronized EEG? To answer this question, we made simultaneous intracellular recordings from two to four neocortical neurons in cat neocortex. We studied how correlated is the occurrence of active and silent states, and how correlated are fluctuations of the membrane potential in pairs of neurons located close one to the other or separated by up to 13 mm. We show that strong long-range correlation of the membrane potential was observed only (i) during the slow oscillation but not during periods without the oscillation, (ii) during periods which included transitions between the states but not during within-the-state periods, and (iii) for the low-frequency (<5 Hz) components of membrane potential fluctuations but not for the higher-frequency components (>10 Hz). In contrast to the neurons located several millimeters one from the other, membrane potential fluctuations in neighboring neurons remain strongly correlated during periods without slow oscillation. We conclude that membrane potential correlation in distant neurons is brought about by synchronous transitions between the states, while activity within the states is largely uncorrelated. The lack of the generalized fine-scale synchronization of membrane potential changes in neurons during the active states of slow oscillation may

Reticulomics: Protein-Protein Interaction Studies with Two Plasmodesmata-Localized Reticulon Family Proteins Identify Binding Partners Enriched at Plasmodesmata, Endoplasmic Reticulum, and the Plasma Membrane.

PubMed

Kriechbaumer, Verena; Botchway, Stanley W; Slade, Susan E; Knox, Kirsten; Frigerio, Lorenzo; Oparka, Karl; Hawes, Chris

2015-11-01

The endoplasmic reticulum (ER) is a ubiquitous organelle that plays roles in secretory protein production, folding, quality control, and lipid biosynthesis. The cortical ER in plants is pleomorphic and structured as a tubular network capable of morphing into flat cisternae, mainly at three-way junctions, and back to tubules. Plant reticulon family proteins (RTNLB) tubulate the ER by dimerization and oligomerization, creating localized ER membrane tensions that result in membrane curvature. Some RTNLB ER-shaping proteins are present in the plasmodesmata (PD) proteome and may contribute to the formation of the desmotubule, the axial ER-derived structure that traverses primary PD. Here, we investigate the binding partners of two PD-resident reticulon proteins, RTNLB3 and RTNLB6, that are located in primary PD at cytokinesis in tobacco (Nicotiana tabacum). Coimmunoprecipitation of green fluorescent protein-tagged RTNLB3 and RTNLB6 followed by mass spectrometry detected a high percentage of known PD-localized proteins as well as plasma membrane proteins with putative membrane-anchoring roles. Förster resonance energy transfer by fluorescence lifetime imaging microscopy assays revealed a highly significant interaction of the detected PD proteins with the bait RTNLB proteins. Our data suggest that RTNLB proteins, in addition to a role in ER modeling, may play important roles in linking the cortical ER to the plasma membrane. © 2015 American Society of Plant Biologists. All Rights Reserved.

Poisoning of mixed matrix membranes by fermentation components in pervaporation of ethanol

USDA-ARS?s Scientific Manuscript database

Pervaporation is an alternative to distillation for recovering ethanol produced by fermentation of grains and biomass. Ethanol-selective mixed matrix membranes of the hydrophobic zeolite ZSM-5 in polydimethylsiloxane (PDMS) have superior performance compared to pure PDMS membranes in pervaporation o...

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

Thermodynamic analysis of membrane fouling in a submerged membrane bioreactor and its implications.

PubMed

Hong, Huachang; Peng, Wei; Zhang, Meijia; Chen, Jianrong; He, Yiming; Wang, Fangyuan; Weng, Xuexiang; Yu, Haiying; Lin, Hongjun

2013-10-01

The thermodynamic interactions between membrane and sludge flocs in a submerged membrane bioreactor (MBR) were investigated. It was found that Lewis acid-base (AB) interaction predominated in the total interactions. The interaction energy composition of membrane-sludge flocs combination was quite similar to that of membrane-bovine serum albumin (BSA) combination, indicating the critical role of proteins in adhesion process. Detailed analysis revealed the existence of a repulsive energy barrier in membrane-foulants interaction. Calculation results demonstrated that small flocs possessed higher attractive interaction energy per unit mass, and therefore adhered to membrane surface more easily as compared to large flocs. Meanwhile, initial sludge adhesion would facilitate the following adhesion due to the reduced repulsive energy barrier. Membrane with high electron donor surface tension component was a favor option for membrane fouling abatement. These findings offered new insights into membrane fouling, and also provided significant implications for fouling control in MBRs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification.

PubMed

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-05-06

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines.

Characterization of anti-leukemia components from Indigo naturalis using comprehensive two-dimensional K562/cell membrane chromatography and in silico target identification

PubMed Central

Wu, Xunxun; Chen, Xiaofei; Dan, Jia; Cao, Yan; Gao, Shouhong; Guo, Zhiying; Zerbe, Philipp; Chai, Yifeng; Diao, Yong; Zhang, Lei

2016-01-01

Traditional Chinese Medicine (TCM) has been developed for thousands of years and has formed an integrated theoretical system based on a large amount of clinical practice. However, essential ingredients in TCM herbs have not been fully identified, and their precise mechanisms and targets are not elucidated. In this study, a new strategy combining comprehensive two-dimensional K562/cell membrane chromatographic system and in silico target identification was established to characterize active components from Indigo naturalis, a famous TCM herb that has been widely used for the treatment of leukemia in China, and their targets. Three active components, indirubin, tryptanthrin and isorhamnetin, were successfully characterized and their anti-leukemia effects were validated by cell viability and cell apoptosis assays. Isorhamnetin, with undefined cancer related targets, was selected for in silico target identification. Proto-oncogene tyrosine-protein kinase (Src) was identified as its membrane target and the dissociation constant (Kd) between Src and isorhamnetin was 3.81 μM. Furthermore, anti-leukemia effects of isorhamnetin were mediated by Src through inducing G2/M cell cycle arrest. The results demonstrated that the integrated strategy could efficiently characterize active components in TCM and their targets, which may bring a new light for a better understanding of the complex mechanism of herbal medicines. PMID:27150638

Mixing, diffusion, and percolation in binary supported membranes containing mixtures of lipids and amphiphilic block copolymers.

PubMed

Gettel, Douglas L; Sanborn, Jeremy; Patel, Mira A; de Hoog, Hans-Peter; Liedberg, Bo; Nallani, Madhavan; Parikh, Atul N

2014-07-23

Substrate-mediated fusion of small polymersomes, derived from mixtures of lipids and amphiphilic block copolymers, produces hybrid, supported planar bilayers at hydrophilic surfaces, monolayers at hydrophobic surfaces, and binary monolayer/bilayer patterns at amphiphilic surfaces, directly responding to local measures of (and variations in) surface free energy. Despite the large thickness mismatch in their hydrophobic cores, the hybrid membranes do not exhibit microscopic phase separation, reflecting irreversible adsorption and limited lateral reorganization of the polymer component. With increasing fluid-phase lipid fraction, these hybrid, supported membranes undergo a fluidity transition, producing a fully percolating fluid lipid phase beyond a critical area fraction, which matches the percolation threshold for the immobile point obstacles. This then suggests that polymer-lipid hybrid membranes might be useful models for studying obstructed diffusion, such as occurs in lipid membranes containing proteins.

Increased localization of APP-C99 in mitochondria-associated ER membranes causes mitochondrial dysfunction in Alzheimer disease.

PubMed

Pera, Marta; Larrea, Delfina; Guardia-Laguarta, Cristina; Montesinos, Jorge; Velasco, Kevin R; Agrawal, Rishi R; Xu, Yimeng; Chan, Robin B; Di Paolo, Gilbert; Mehler, Mark F; Perumal, Geoffrey S; Macaluso, Frank P; Freyberg, Zachary Z; Acin-Perez, Rebeca; Enriquez, Jose Antonio; Schon, Eric A; Area-Gomez, Estela

2017-11-15

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by γ-secretase to generate the β-amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Sorting of amphiphile membrane components in curvature and composition gradients

NASA Astrophysics Data System (ADS)

Tian, Aiwei

Phase and shape heterogeneities in biomembranes are of functional importance. However, it is difficult to elucidate the roles membrane heterogeneities play in maintaining cellular function due to the complexity of biomembranes. Therefore, investigations of phase behavior and composition/curvature coupling in lipid and polymer model membranes offer some advantages. In this thesis, phase properties in lipid and polymer giant vesicles were studied. Line tension at the fluid/fluid phase boundary of giant lipid unilamellar vesicles was determined directly by micropipette aspiration, and found to be composition-dependent. Dynamics of calcium-induced domains within polyanionic vesicles subject to chemical stimuli were investigated, which revealed the strength of molecular interaction and suggested applications in triggered delivery. In addition, curvature sorting of lipids and proteins was examined. Lipid membrane tethers were pulled from giant unilamellar vesicles using two micropipettes and a bead. Tether radius can be controlled and measured in this system. By examining fluorescence intensity of labeled molecules as a function of curvature, we found that DiI dyes (lipid analogues with spontaneous curvatures) had no curvature preference down to radii of 10 nm. Theoretical calculation predicted that the distribution of small lipids was dominated by entropy instead of bending energy. However protein Cholera toxin subunit B was efficiently sorted away from the high positive curvature due to its negative spontaneous curvature. Bending stiffness was determined to decrease as curvature increased in homogeneous membranes with ternary lipid mixtures near a critical consulate point, revealing the strong preferential intermolecular interactions of such mixtures. In addition, diffusion controlled domain growth was observed in tethers pulled from phase-separated vesicles, which provides a new dynamic sorting principle for lipids and proteins in curvature gradients.

CALCIUM BINDING TO INTESTINAL MEMBRANES

PubMed Central

Oschman, James L.; Wall, Betty J.

1972-01-01

Flame photometry reveals that glutaraldehyde and buffer solutions in routine use for electron microscopy contain varying amounts of calcium. The presence of electron-opaque deposits adjacent to membranes in a variety of tissues can be correlated with the presence of calcium in the fixative. In insect intestine (midgut), deposits occur adjacent to apical and lateral plasma membranes. The deposits are particularly evident in tissues fixed in glutaraldehyde without postosmication. They are also observed in osmicated tissue if calcium is added to wash and osmium solutions. Deposits are absent when calcium-free fixatives are used, but are present when traces of CaCl2 (as low as 5 x 10-5 M) are added. The deposits occur at regular intervals along junctional membranes, providing images strikingly similar to those obtained by other workers who have used pyroantimonate in an effort to localize sodium. Other divalent cations (Mg++, Sr++, Ba++, Mn++, Fe++) appear to substitute for calcium, while sodium, potassium, lanthanum, and mercury do not. After postfixing with osmium with calcium added, the deposits can be resolved as patches along the inner leaflet of apical and lateral plasma membranes. The dense regions may thus localize membrane constituents that bind calcium. The results are discussed in relation to the role of calcium in control of cell-to-cell communication, intestinal calcium uptake, and the pyroantimonate technique for ion localization. PMID:4569411

REDOR solid-state NMR as a probe of the membrane locations of membrane-associated peptides and proteins

NASA Astrophysics Data System (ADS)

Jia, Lihui; Liang, Shuang; Sackett, Kelly; Xie, Li; Ghosh, Ujjayini; Weliky, David P.

2015-04-01

Rotational-echo double-resonance (REDOR) solid-state NMR is applied to probe the membrane locations of specific residues of membrane proteins. Couplings are measured between protein 13CO nuclei and membrane lipid or cholesterol 2H and 31P nuclei. Specific 13CO labeling is used to enable unambiguous assignment and 2H labeling covers a small region of the lipid or cholesterol molecule. The 13CO-31P and 13CO-2H REDOR respectively probe proximity to the membrane headgroup region and proximity to specific insertion depths within the membrane hydrocarbon core. One strength of the REDOR approach is use of chemically-native proteins and membrane components. The conventional REDOR pulse sequence with 100 kHz 2H π pulses is robust with respect to the 2H quadrupolar anisotropy. The 2H T1's are comparable to the longer dephasing times (τ's) and this leads to exponential rather than sigmoidal REDOR buildups. The 13CO-2H buildups are well-fitted to A × (1 - e-γτ) where A and γ are fitting parameters that are correlated as the fraction of molecules (A) with effective 13CO-2H coupling d = 3γ/2. The REDOR approach is applied to probe the membrane locations of the "fusion peptide" regions of the HIV gp41 and influenza virus hemagglutinin proteins which both catalyze joining of the viral and host cell membranes during initial infection of the cell. The HIV fusion peptide forms an intermolecular antiparallel β sheet and the REDOR data support major deeply-inserted and minor shallowly-inserted molecular populations. A significant fraction of the influenza fusion peptide molecules form a tight hairpin with antiparallel N- and C-α helices and the REDOR data support a single peptide population with a deeply-inserted N-helix. The shared feature of deep insertion of the β and α fusion peptide structures may be relevant for fusion catalysis via the resultant local perturbation of the membrane bilayer. Future applications of the REDOR approach may include samples that contain cell

Influence of local-field corrections on Thomson scattering in collision-dominated two-component plasmas.

PubMed

Fortmann, Carsten; Wierling, August; Röpke, Gerd

2010-02-01

The dynamic structure factor, which determines the Thomson scattering spectrum, is calculated via an extended Mermin approach. It incorporates the dynamical collision frequency as well as the local-field correction factor. This allows to study systematically the impact of electron-ion collisions as well as electron-electron correlations due to degeneracy and short-range interaction on the characteristics of the Thomson scattering signal. As such, the plasmon dispersion and damping width is calculated for a two-component plasma, where the electron subsystem is completely degenerate. Strong deviations of the plasmon resonance position due to the electron-electron correlations are observed at increasing Brueckner parameters r(s). These results are of paramount importance for the interpretation of collective Thomson scattering spectra, as the determination of the free electron density from the plasmon resonance position requires a precise theory of the plasmon dispersion. Implications due to different approximations for the electron-electron correlation, i.e., different forms of the one-component local-field correction, are discussed.

Performance of Water Recirculation Loop Maintenance Components for the Advanced Spacesuit Water Membrane Evaporator

NASA Technical Reports Server (NTRS)

Rector, Tony; Peyton, Barbara M.; Steele, John W.; Makinen, Janice; Bue, Grant C.; Campbell, Colin

2014-01-01

Water loop maintenance components to maintain the water quality of the Advanced Spacesuit Water Membrane Evaporation (SWME) water recirculation loop have undergone a comparative performance evaluation with a second SWME water recirculation loop with no water quality maintenance. Results show the benefits of periodic water maintenance. The SWME is a heat rejection device under development at the NASA Johnson Space Center to perform thermal control for advanced spacesuits. One advantage to this technology is the potential for a significantly greater degree of tolerance to contamination when compared to the existing Sublimator technology. The driver for the evaluation of water recirculation maintenance components was to further enhance this advantage through the leveraging of fluid loop management lessons learned from the International Space Station (ISS). A bed design that was developed for a UTAS military application, and considered for a potential ISS application with the Urine Processor Assembly, provided a low pressure drop means for water maintenance in a recirculation loop. The bed design is coupled with high capacity ion exchange resins, organic adsorbents, and a cyclic methodology developed for the Extravehicular Mobility Unit (EMU) Transport Water loop. The maintenance cycle included the use of a biocide delivery component developed for ISS to introduce a biocide in a microgravity compatible manner for the Internal Active Thermal Control System (IATCS). The leveraging of these water maintenance technologies to the SWME recirculation loop is a unique demonstration of applying the valuable lessons learned on the ISS to the next generation of manned spaceflight Environmental Control and Life Support System (ECLSS) hardware.

Performance of Water Recirculation Loop Maintenance Components for the Advanced Spacesuit Water Membrane Evaporator

NASA Technical Reports Server (NTRS)

Rector, Tony; Peyton, Barbara M.; Steele, John W.; Makinen, Janice; Bue, Grant C.; Campbell, Colin

2014-01-01

Water loop maintenance components to maintain the water quality of the Advanced Spacesuit Water Membrane Evaporation (SWME) water recirculation loop have undergone a comparative performance evaluation with a recirculating control loop which had no water quality maintenance. Results show that periodic water maintenance can improve performance of the SWME. The SWME is a heat rejection device under development at the NASA Johnson Space Center to perform thermal control for advanced spacesuits. One advantage of this technology is the potential for a significantly greater degree of tolerance to contamination when compared to the existing sublimator technology. The driver for the evaluation of water recirculation maintenance components was to enhance the robustness of the SWME through the leveraging of fluid loop management lessons learned from the International Space Station (ISS). A patented bed design that was developed for a United Technologies Aerospace System military application provided a low pressure drop means for water maintenance in the SWME recirculation loop. The bed design is coupled with high capacity ion exchange resins, organic adsorbents, and a cyclic methodology developed for the Extravehicular Mobility Unit (EMU) Transport Water loop. The maintenance cycle included the use of a biocide delivery component developed for the ISS to introduce a biocide in a microgravity compatible manner for the Internal Active Thermal Control System (IATCS). The leveraging of these water maintenance technologies to the SWME recirculation loop is a unique demonstration of applying the valuable lessons learned on the ISS to the next generation of manned spaceflight Environmental Control and Life Support System (ECLSS) hardware.

Performance of Water Recirculation Loop Maintentance Components for the Advanced Spacesuit Water Membrane Evaporator

NASA Technical Reports Server (NTRS)

Rector, Tony; Peyton, Barbara; Steele, John W.; Bue, Grant C.; Campbell, Colin; Makinen, Janice

2014-01-01

Water loop maintenance components to maintain the water quality of the Advanced Spacesuit Water Membrane Evaporation (SWME) water recirculation loop have undergone a comparative performance evaluation with a second SWME water recirculation loop with no water quality maintenance. Results show the benefits of periodic water maintenance. The SWME is a heat rejection device under development at the NASA Johnson Space Center to perform thermal control for advanced spacesuits. One advantage to this technology is the potential for a significantly greater degree of tolerance to contamination when compared to the existing Sublimator technology. The driver for the evaluation of water recirculation maintenance components was to further enhance this advantage through the leveraging of fluid loop management lessonslearned from the International Space Station (ISS). A bed design that was developed for a UTAS military application, and considered for a potential ISS application with the Urine Processor Assembly, provided a low pressure drop means for water maintenance in a recirculation loop. The bed design is coupled with high capacity ion exchange resins, organic adsorbents, and a cyclic methodology developed for the Extravehicular Mobility Unit (EMU) Transport Water loop. The maintenance cycle included the use of a biocide delivery component developed for ISS to introduce a biocide in a microgravity-compatible manner for the Internal Active Thermal Control System (IATCS). The leveraging of these water maintenance technologies to the SWME recirculation loop is a unique demonstration of applying the valuable lessons learned on the ISS to the next generation of manned spaceflight Environmental Control and Life Support System (ECLSS) hardware.

Localization and Characterization of Photosystem II in Grana and Stroma Lamellae 1

PubMed Central

Armond, Paul A.; Arntzen, Charles J.

1977-01-01

Attempts have been made to identify intramembranous particles observed in freeze-fracture electron microscopy as specific functional components of the membrane. The intramembranous particles of the exoplasmic fracture (EF) face of freeze-fractured pea (Pisum sativum) chloroplast lamellae are nonuniformly distributed along the membrane. Approximately 20% of the particles are in unpaired membrane regions whereas 80% are localized in regions of stacked lamellae (grana partitions). The EF particles within the grana regions of the chloroplast membrane are of a larger average size than those in stroma lamellae. Photosystem II activity of isolated stroma lamellae is about 20 to 25% of that of grana-enriched membrane fragments when measured at high light intensities. The photosystem II activity of stroma lamellae requires higher light intensities for attainment of maximal rates than does that of grana membranes. Lactoperoxidase-catalyzed iodination of stacked chloroplast lamellae was used to demonstrate that 75 to 80% of all photosystem II centers are localized in grana partition regions. The data presented support the concept that the intramembranous particles of the EF face visualized on freeze-fractured chloroplast lamellae represent a central photosystem II reaction center complex plus associated light-harvesting chlorophyll protein. The fact that the EF particles of stroma lamellae are smaller than those of grana regions can be directly correlated to the presence of photosystem II units with small antennae chlorophyll assemblies in stroma lamellae. Images PMID:16659861

One-dimensional nonlinear elastodynamic models and their local conservation laws with applications to biological membranes.

PubMed

Cheviakov, A F; Ganghoffer, J-F

2016-05-01

The framework of incompressible nonlinear hyperelasticity and viscoelasticity is applied to the derivation of one-dimensional models of nonlinear wave propagation in fiber-reinforced elastic solids. Equivalence transformations are used to simplify the resulting wave equations and to reduce the number of parameters. Local conservation laws and global conserved quantities of the models are systematically computed and discussed, along with other related mathematical properties. Sample numerical solutions are presented. The models considered in the paper are appropriate for the mathematical description of certain aspects of the behavior of biological membranes and similar structures. Copyright © 2015 Elsevier Ltd. All rights reserved.

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

PubMed Central

Radhakrishna, Harish; Donaldson, Julie G.

1997-01-01

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

Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

PubMed

Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

2017-08-01

While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

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

PubMed

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

2015-12-15

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

Membrane-Mediated Cooperativity of Proteins

NASA Astrophysics Data System (ADS)

Weikl, Thomas R.

2018-04-01

Besides direct protein-protein interactions, indirect interactions mediated by membranes play an important role for the assembly and cooperative function of proteins in membrane shaping and adhesion. The intricate shapes of biological membranes are generated by proteins that locally induce membrane curvature. Indirect curvature-mediated interactions between these proteins arise because the proteins jointly affect the bending energy of the membranes. These curvature-mediated interactions are attractive for crescent-shaped proteins and are a driving force in the assembly of the proteins during membrane tubulation. Membrane adhesion results from the binding of receptor and ligand proteins that are anchored in the apposing membranes. The binding of these proteins strongly depends on nanoscale shape fluctuations of the membranes, leading to a fluctuation-mediated binding cooperativity. A length mismatch between receptor-ligand complexes in membrane adhesion zones causes repulsive curvature-mediated interactions that are a driving force for the length-based segregation of proteins during membrane adhesion.

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.

Linking Findings in Microfluidics to Membrane Emulsification Process Design: The Importance of Wettability and Component Interactions with Interfaces

PubMed Central

Schroën, Karin; Ferrando, Montse; de Lamo-Castellví, Silvia; Sahin, Sami; Güell, Carme

2016-01-01

In microfluidics and other microstructured devices, wettability changes, as a result of component interactions with the solid wall, can have dramatic effects. In emulsion separation and emulsification applications, the desired behavior can even be completely lost. Wettability changes also occur in one phase systems, but the effect is much more far-reaching when using two-phase systems. For microfluidic emulsification devices, this can be elegantly demonstrated and quantified for EDGE (Edge-base Droplet GEneration) devices that have a specific behavior that allows us to distinguish between surfactant and liquid interactions with the solid surface. Based on these findings, design rules can be defined for emulsification with any micro-structured emulsification device, such as direct and premix membrane emulsification. In general, it can be concluded that mostly surface interactions increase the contact angle toward 90°, either through the surfactant, or the oil that is used. This leads to poor process stability, and very limited pressure ranges at which small droplets can be made in microfluidic systems, and cross-flow membrane emulsification. In a limited number of cases, surface interactions can also lead to lower contact angles, thereby increasing the operational stability. This paper concludes with a guideline that can be used to come to the appropriate combination of membrane construction material (or any micro-structured device), surfactants and liquids, in combination with process conditions. PMID:27187484

DSC and EPR investigations on effects of cholesterol component on molecular interactions between paclitaxel and phospholipid within lipid bilayer membrane.

PubMed

Zhao, Lingyun; Feng, Si-Shen; Kocherginsky, Nikolai; Kostetski, Iouri

2007-06-29

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR) were applied to investigate effects of cholesterol component on molecular interactions between paclitaxel, which is one of the best antineoplastic agents found from nature, and dipalmitoylphosphatidylcholine (DPPC) within lipid bilayer vesicles (liposomes), which could also be used as a model cell membrane. DSC analysis showed that incorporation of paclitaxel into the DPPC bilayer causes a reduction in the cooperativity of bilayer phase transition, leading to a looser and more flexible bilayer structure. Including cholesterol component in the DPPC/paclitaxel mixed bilayer can facilitate the molecular interaction between paclitaxel and lipid and make the tertiary system more stable. EPR analysis demonstrated that both of paclitaxel and cholesterol have fluidization effect on the DPPC bilayer membranes although cholesterol has more significant effect than paclitaxel does. The reduction kinetics of nitroxides by ascorbic acid showed that paclitaxel can inhibit the reaction by blocking the diffusion of either the ascorbic acid or nitroxide molecules since the reaction is tested to be a first order one. Cholesterol can remarkably increase the reduction reaction speed. This research may provide useful information for optimizing liposomal formulation of the drug as well as for understanding the pharmacology of paclitaxel.

Membrane localization of insulin receptor substrate-2 (IRS-2) is associated with decreased overall survival in breast cancer

PubMed Central

Clark, Jennifer L.; Dresser, Karen; Hsieh, Chung-Cheng; Sabel, Michael; Kleer, Celina G.; Khan, Ashraf

2011-01-01

Recent studies have identified a role for insulin receptor substrate-2 (IRS-2) in promoting motility and metastasis in breast cancer. However, no published studies to date have examined IRS-2 expression in human breast tumors. We examined IRS-2 expression by immunohistochemistry (IHC) in normal breast tissue, benign breast lesions, and malignant breast tumors from the institutional pathology archives and a tumor microarray from a separate institution. Three distinct IRS-2 staining patterns were noted: diffusely cytoplasmic, punctate cytoplasmic, and localized to the cell membrane. The individual and pooled datasets were analyzed for associations of IRS-2 staining pattern with core clinical parameters and clinical outcomes. Univariate analysis revealed a trend toward decreased overall survival (OS) with IRS-2 membrane staining, and this association became significant upon multivariate analysis (P = 0.01). In progesterone receptor negative (PR−) tumors, in particular, IRS-2 staining at the membrane correlated with significantly worse OS than other IRS-2 staining patterns (P < 0.001). When PR status and IRS-2 staining pattern were evaluated in combination, PR− tumors with IRS-2 at the membrane were associated with a significantly decreased OS when compared with all other combinations (P = 0.002). Evaluation of IRS-2 staining patterns could potentially be used to identify patients with PR− tumors who would most benefit from aggressive treatment. PMID:21258861

Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

PubMed

Liu, Jianping; Hayashi, Kyoko; Matsuoka, Ken

2015-01-01

S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

Theoretical investigation of local proton conductance in the proton exchange membranes

NASA Astrophysics Data System (ADS)

Singh, Raman K.; Tsuneda, Takao; Miyatake, Kenji; Watanabe, Masahiro

2014-07-01

The hydrated structures of the proton exchange membranes were theoretically investigated using long-range corrected density functional theory to make clear why perfluorinated polymer membrane Nafion is superior to other membranes in the proton conductivity at low humidity. For exploring the possibility of the proton conductance in the vehicle mechanism with low hydration numbers, we examined the relay model of protonated water clusters between the sulfonic acid groups in Nafion and concluded that this relay model may contribute to the high proton conductivity of Nafion with less-hydrated sulfonic acid groups.

Proteasome inhibitor (MG-132) treatment of mdx mice rescues the expression and membrane localization of dystrophin and dystrophin-associated proteins.

PubMed

Bonuccelli, Gloria; Sotgia, Federica; Schubert, William; Park, David S; Frank, Philippe G; Woodman, Scott E; Insabato, Luigi; Cammer, Michael; Minetti, Carlo; Lisanti, Michael P

2003-10-01

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is absent in the skeletal muscle of DMD patients and mdx mice. At the plasma membrane of skeletal muscle fibers, dystrophin associates with a multimeric protein complex, termed the dystrophin-glycoprotein complex (DGC). Protein members of this complex are normally absent or greatly reduced in dystrophin-deficient skeletal muscle fibers, and are thought to undergo degradation through an unknown pathway. As such, we reasoned that inhibition of the proteasomal degradation pathway might rescue the expression and subcellular localization of dystrophin-associated proteins. To test this hypothesis, we treated mdx mice with the well-characterized proteasomal inhibitor MG-132. First, we locally injected MG-132 into the gastrocnemius muscle, and observed the outcome after 24 hours. Next, we performed systemic treatment using an osmotic pump that allowed us to deliver different concentrations of the proteasomal inhibitor, over an 8-day period. By immunofluorescence and Western blot analysis, we show that administration of the proteasomal inhibitor MG-132 effectively rescues the expression levels and plasma membrane localization of dystrophin, beta-dystroglycan, alpha-dystroglycan, and alpha-sarcoglycan in skeletal muscle fibers from mdx mice. Furthermore, we show that systemic treatment with the proteasomal inhibitor 1) reduces muscle membrane damage, as revealed by vital staining (with Evans blue dye) of the diaphragm and gastrocnemius muscle isolated from treated mdx mice, and 2) ameliorates the histopathological signs of muscular dystrophy, as judged by hematoxylin and eosin staining of muscle biopsies taken from treated mdx mice. Thus, the current study opens new and important avenues in our understanding of the pathogenesis of DMD. Most importantly, these new findings may have clinical implications for the pharmacological treatment of patients with DMD.

Effect of biological and coagulation pre-treatments to control organic and biofouling potential components of ultrafiltration membrane in the treatment of lake water.

PubMed

Pramanik, Biplob Kumar; Kajol, Annaduzzaman; Suja, Fatihah; Md Zain, Shahrom

2017-03-01

Biological aerated filter (BAF), sand filtration (SF), alum and Moringa oleifera coagulation were investigated as a pre-treatment for reducing the organic and biofouling potential component of an ultrafiltration (UF) membrane in the treatment of lake water. The carbohydrate content was mainly responsible for reversible fouling of the UF membrane compared to protein or dissolved organic carbon (DOC) content. All pre-treatment could effectively reduce these contents and led to improve the UF filterability. Both BAF and SF markedly led to improvement in flux than coagulation processes, and alum gave greater flux than M. oleifera. This was attributed to the effective removal and/or breakdown of high molecular weight (MW) organics by biofilters. BAF led to greater improvement in flux than SF, due to greater breakdown of high MW organics, and this was also confirmed by the attenuated total reflection-Fourier transform infrared spectroscopy analysis. Coagulation processes were ineffective in removing biofouling potential components, whereas both biofilters were very effective as shown by the reduction of low MW organics, biodegradable dissolved organic carbon and assimilable organic carbon contents. This study demonstrated the potential of biological pre-treatments for reducing organic and biofouling potential component and thus improving flux for the UF of lake water treatment.

Plasma membrane repair in plants.

PubMed

Schapire, Arnaldo L; Valpuesta, Victoriano; Botella, Miguel A

2009-12-01

Resealing is the membrane-repair process that enables cells to survive disruption, preventing the loss of irreplaceable cell types and eliminating the cost of replacing injured cells. Given that failure in the resealing process in animal cells causes diverse types of muscular dystrophy, plasma membrane repair has been extensively studied in these systems. Animal proteins with Ca(2+)-binding domains such as synaptotagmins and dysferlin mediate Ca(2+)-dependent exocytosis to repair plasma membranes after mechanical damage. Until recently, no components or proof for membrane repair mechanisms have been discovered in plants. However, Arabidopsis SYT1 is now the first plant synaptotagmin demonstrated to participate in Ca(2+)-dependent repair of membranes. This suggests a conservation of membrane repair mechanisms between animal and plant cells.

Immunofluorescence localization of dissociation supernatant and extracellular matrix components in Lytechinus pictus sectioned embryos. M.S. Thesis

NASA Technical Reports Server (NTRS)

Garciaflack, Ana Leticia

1988-01-01

Indirect immunofluorescence was used to localize specific extracellular components in embryos of the sea urchin Lytechinus pictus. Hyalin and S2 (a group of components found in the disaggregation supernatant from Strongylocentrotus purpuratus blastulae) were uniformly present at all stages (unfertilized up to 32 hr) except hyalin could not be detected at the 12 hour early blastula stage. Laminin was found in 16 cell, 32 cell, 6 hour, 18 hour, 24 hour, and 32 hour stages, with especially bright fluorescence at 18 hours. Collagen I was present at all stages (freshly fertilized up to 32 hour) except little was detected at 12 hours. Fibronectin was uniformly present in blastocoelar fibers stained with anto-collagen I and anti-fibronectin. These results were compared with those for S. purpuratus to produce an overview of the localization of specific extracellular matrix components during development of two species of sea urchins. The results set the stage for future studies that will examine the function of these components at the various developmental stages.

Quantum field theory with infinite component local fields as an alternative to the string theories

NASA Astrophysics Data System (ADS)

Krasnikov, N. V.

1987-09-01

We show that the introduction of the infinite component local fields with higher-order derivatives in the interaction makes the theory completely ultraviolet finite. For the γ5-anomalous theories the introduction of the infinite component field makes the theory renormalizable or even superrenormalizable. I am indebted to J. Ambjōrn, P. Di Vecchia, H.B. Nielsen and L. Rozhansky for useful discussions. It is a pleasure to thank the Niels Bohr Institute (Copenhagen) where this work was completed for kind hospitality.

Systematically Ranking the Tightness of Membrane Association for Peripheral Membrane Proteins (PMPs)*

PubMed Central

Gao, Liyan; Ge, Haitao; Huang, Xiahe; Liu, Kehui; Zhang, Yuanya; Xu, Wu; Wang, Yingchun

2015-01-01

Large-scale quantitative evaluation of the tightness of membrane association for nontransmembrane proteins is important for identifying true peripheral membrane proteins with functional significance. Herein, we simultaneously ranked more than 1000 proteins of the photosynthetic model organism Synechocystis sp. PCC 6803 for their relative tightness of membrane association using a proteomic approach. Using multiple precisely ranked and experimentally verified peripheral subunits of photosynthetic protein complexes as the landmarks, we found that proteins involved in two-component signal transduction systems and transporters are overall tightly associated with the membranes, whereas the associations of ribosomal proteins are much weaker. Moreover, we found that hypothetical proteins containing the same domains generally have similar tightness. This work provided a global view of the structural organization of the membrane proteome with respect to divergent functions, and built the foundation for future investigation of the dynamic membrane proteome reorganization in response to different environmental or internal stimuli. PMID:25505158

A New Polysulfone Membrane Dialyzer, NV, with Low-Fouling and Antithrombotic Properties.

PubMed

Oshihara, Wataru; Ueno, Yoshiyuki; Fujieda, Hiroaki

2017-01-01

The biggest problem in routine hemodialysis therapy is possibly the blood pressure fall experienced by patients during dialysis. In contrast, in medium- and long-term hemodialysis therapy, the main problem might be deterioration of arteriosclerosis because of medial calcification associated with dialysis vintage. Both problems are caused by an autonomic imbalance or structural change in the blood vessels. Inflammation due to extracorporeal blood circulation is another possible cause. This inflammation is considered to cause platelets activated by contact and adherence with the membrane surface to aggregate with white blood cells and attack the endothelium of the blood vessels. Therefore, we tried to develop a new membrane with no adsorption and no platelet activation. Polysulfone (PS) membranes with polyvinylpyrrolidone (PVP) as a hydrophilic agent are widely used in dialysis, but blood components adhere to the membrane surface. We developed a new dialyzer, NV, by localizing a new hydrophilic polymer onto the inner surface of a hollow-fiber membrane composed of PS and PVP. The number of platelets that adhered to the NV membrane surface drastically decreased to 0.9% of that with the conventional PS dialysis membrane. We also confirmed the mechanism by which NV realizes clinical improvements in blood pressure drops and inflammation during dialysis, and verified its clinical appeal. Key Messages: The new membrane NV, which inhibits platelet adhesion and is compatible with blood vessels, is clinically beneficial. © 2017 S. Karger AG, Basel.

The Role of Electron Microscopy in Studying the Continuum of Changes in Membranous Structures during Poliovirus Infection

PubMed Central

Rossignol, Evan D.; Yang, Jie E.; Bullitt, Esther

2015-01-01

Replication of the poliovirus genome is localized to cytoplasmic replication factories that are fashioned out of a mixture of viral proteins, scavenged cellular components, and new components that are synthesized within the cell due to viral manipulation/up-regulation of protein and phospholipid synthesis. These membranous replication factories are quite complex, and include markers from multiple cytoplasmic cellular organelles. This review focuses on the role of electron microscopy in advancing our understanding of poliovirus RNA replication factories. Structural data from the literature provide the basis for interpreting a wide range of biochemical studies that have been published on virus-induced lipid biosynthesis. In combination, structural and biochemical experiments elucidate the dramatic membrane remodeling that is a hallmark of poliovirus infection. Temporal and spatial membrane modifications throughout the infection cycle are discussed. Early electron microscopy studies of morphological changes following viral infection are re-considered in light of more recent data on viral manipulation of lipid and protein biosynthesis. These data suggest the existence of distinct subcellular vesicle populations, each of which serves specialized roles in poliovirus replication processes. PMID:26473912

Polar transport in plants mediated by membrane transporters: focus on mechanisms of polar auxin transport.

PubMed

Naramoto, Satoshi

2017-12-01

Directional cell-to-cell transport of functional molecules, called polar transport, enables plants to sense and respond to developmental and environmental signals. Transporters that localize to plasma membranes (PMs) in a polar manner are key components of these systems. PIN-FORMED (PIN) auxin efflux carriers, which are the most studied polar-localized PM proteins, are implicated in the polar transport of auxin that in turn regulates plant development and tropic growth. In this review, the regulatory mechanisms underlying polar localization of PINs, control of auxin efflux activity, and PIN abundance at PMs are considered. Up to date information on polar-localized nutrient transporters that regulate directional nutrient movement from soil into the root vasculature is also discussed. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells*

PubMed Central

An, Hyun Joo; Gip, Phung; Kim, Jaehan; Wu, Shuai; Park, Kun Wook; McVaugh, Cheryl T.; Schaffer, David V.; Bertozzi, Carolyn R.; Lebrilla, Carlito B.

2012-01-01

Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine

Perspective of membrane technology in dairy industry: a review.

PubMed

Kumar, Pavan; Sharma, Neelesh; Ranjan, Rajeev; Kumar, Sunil; Bhat, Z F; Jeong, Dong Kee

2013-09-01

Membrane technology has revolutionized the dairy sector. Different types of membranes are used in the industry for various purposes like extending the shelf life of milk without exposure to heat treatment, standardization of the major components of milk for tailoring new products as well increasing yield and quality of the dairy products, and concentrating, fractionation and purification of milk components especially valuable milk proteins in their natural state. In the cheese industry, membranes increase the yield and quality of cheese and control the whey volume, by concentrating the cheese milk. With the advancement of newer technology in membrane processes, it is possible to recover growth factor from whey. With the introduction of superior quality membranes as well as newer technology, the major limitation of membranes, fouling or blockage has been overcome to a greater extent.

Non-Brownian diffusion in lipid membranes: Experiments and simulations.

PubMed

Metzler, R; Jeon, J-H; Cherstvy, A G

2016-10-01

The dynamics of constituents and the surface response of cellular membranes-also in connection to the binding of various particles and macromolecules to the membrane-are still a matter of controversy in the membrane biophysics community, particularly with respect to crowded membranes of living biological cells. We here put into perspective recent single particle tracking experiments in the plasma membranes of living cells and supercomputing studies of lipid bilayer model membranes with and without protein crowding. Special emphasis is put on the observation of anomalous, non-Brownian diffusion of both lipid molecules and proteins embedded in the lipid bilayer. While single component, pure lipid bilayers in simulations exhibit only transient anomalous diffusion of lipid molecules on nanosecond time scales, the persistence of anomalous diffusion becomes significantly longer ranged on the addition of disorder-through the addition of cholesterol or proteins-and on passing of the membrane lipids to the gel phase. Concurrently, experiments demonstrate the anomalous diffusion of membrane embedded proteins up to macroscopic time scales in the minute time range. Particular emphasis will be put on the physical character of the anomalous diffusion, in particular, the occurrence of ageing observed in the experiments-the effective diffusivity of the measured particles is a decreasing function of time. Moreover, we present results for the time dependent local scaling exponent of the mean squared displacement of the monitored particles. Recent results finding deviations from the commonly assumed Gaussian diffusion patterns in protein crowded membranes are reported. The properties of the displacement autocorrelation function of the lipid molecules are discussed in the light of their appropriate physical anomalous diffusion models, both for non-crowded and crowded membranes. In the last part of this review we address the upcoming field of membrane distortion by elongated membrane

Overexpression of a Plasma Membrane-Localized SbSRP-Like Protein Enhances Salinity and Osmotic Stress Tolerance in Transgenic Tobacco

PubMed Central

Udawat, Pushpika; Jha, Rajesh K.; Mishra, Avinash; Jha, Bhavanath

2017-01-01

An obligate halophyte, Salicornia brachiata grows in salt marshes and is considered to be a potential resource of salt- and drought-responsive genes. It is important to develop an understanding of the mechanisms behind enhanced salt tolerance. To increase this understanding, a novel SbSRP gene was cloned, characterized, over-expressed, and functionally validated in the model plant Nicotiana tabacum. The genome of the halophyte S. brachiata contains two homologs of an intronless SbSRP gene of 1,262 bp in length that encodes for a stress-related protein. An in vivo localization study confirmed that SbSRP is localized on the plasma membrane. Transgenic tobacco plants (T1) that constitutively over-express the SbSRP gene showed improved salinity and osmotic stress tolerance. In comparison to Wild Type (WT) and Vector Control (VC) plants, transgenic lines showed elevated relative water and chlorophyll content, lower malondialdehyde content, lower electrolyte leakage and higher accumulation of proline, free amino acids, sugars, polyphenols, and starch under abiotic stress treatments. Furthermore, a lower build-up of H2O2 content and superoxide-radicals was found in transgenic lines compared to WT and VC plants under stress conditions. Transcript expression of Nt-APX (ascorbate peroxidase), Nt-CAT (catalase), Nt-SOD (superoxide dismutase), Nt-DREB (dehydration responsive element binding factor), and Nt-AP2 (apetala2) genes was higher in transgenic lines under stress compared to WT and VC plants. The results suggested that overexpression of membrane-localized SbSRP mitigates salt and osmotic stress in the transgenic tobacco plant. It was hypothesized that SbSRP can be a transporter protein to transmit the environmental stimuli downward through the plasma membrane. However, a detailed study is required to ascertain its exact role in the abiotic stress tolerance mechanism. Overall, SbSRP is a potential candidate to be used for engineering salt and osmotic tolerance in crops

Mitochondrial protein import: Mia40 facilitates Tim22 translocation into the inner membrane of mitochondria.

PubMed

Wrobel, Lidia; Trojanowska, Agata; Sztolsztener, Malgorzata E; Chacinska, Agnieszka

2013-03-01

The mitochondrial intermembrane space assembly (MIA) pathway is generally considered to be dedicated to the redox-dependent import and biogenesis of proteins localized to the intermembrane space of mitochondria. The oxidoreductase Mia40 is a central component of the pathway responsible for the transfer of disulfide bonds to intermembrane space precursor proteins, causing their oxidative folding. Here we present the first evidence that the function of Mia40 is not restricted to the transport and oxidative folding of intermembrane space proteins. We identify Tim22, a multispanning membrane protein and core component of the TIM22 translocase of inner membrane, as a protein with cysteine residues undergoing oxidation during Tim22 biogenesis. We show that Mia40 is involved in the biogenesis and complex assembly of Tim22. Tim22 forms a disulfide-bonded intermediate with Mia40 upon import into mitochondria. Of interest, Mia40 binds the Tim22 precursor also via noncovalent interactions. We propose that Mia40 not only is responsible for disulfide bond formation, but also assists the Tim22 protein in its integration into the inner membrane of mitochondria.

Unmasking local activity within local field potentials (LFPs) by removing distal electrical signals using independent component analysis.

PubMed

Whitmore, Nathan W; Lin, Shih-Chieh

2016-05-15

Local field potentials (LFPs) are commonly thought to reflect the aggregate dynamics in local neural circuits around recording electrodes. However, we show that when LFPs are recorded in awake behaving animals against a distal reference on the skull as commonly practiced, LFPs are significantly contaminated by non-local and non-neural sources arising from the reference electrode and from movement-related noise. In a data set with simultaneously recorded LFPs and electroencephalograms (EEGs) across multiple brain regions while rats perform an auditory oddball task, we used independent component analysis (ICA) to identify signals arising from electrical reference and from volume-conducted noise based on their distributed spatial pattern across multiple electrodes and distinct power spectral features. These sources of distal electrical signals collectively accounted for 23-77% of total variance in unprocessed LFPs, as well as most of the gamma oscillation responses to the target stimulus in EEGs. Gamma oscillation power was concentrated in volume-conducted noise and was tightly coupled with the onset of licking behavior, suggesting a likely origin of muscle activity associated with body movement or orofacial movement. The removal of distal signal contamination also selectively reduced correlations of LFP/EEG signals between distant brain regions but not within the same region. Finally, the removal of contamination from distal electrical signals preserved an event-related potential (ERP) response to auditory stimuli in the frontal cortex and also increased the coupling between the frontal ERP amplitude and neuronal activity in the basal forebrain, supporting the conclusion that removing distal electrical signals unmasked local activity within LFPs. Together, these results highlight the significant contamination of LFPs by distal electrical signals and caution against the straightforward interpretation of unprocessed LFPs. Our results provide a principled approach to

Unmasking local activity within local field potentials (LFPs) by removing distal electrical signals using independent component analysis

PubMed Central

Whitmore, Nathan W.; Lin, Shih-Chieh

2016-01-01

Local field potentials (LFPs) are commonly thought to reflect the aggregate dynamics in local neural circuits around recording electrodes. However, we show that when LFPs are recorded in awake behaving animals against a distal reference on the skull as commonly practiced, LFPs are significantly contaminated by non-local and non-neural sources arising from the reference electrode and from movement-related noise. In a data set with simultaneously recorded LFPs and electroencephalograms (EEGs) across multiple brain regions while rats perform an auditory oddball task, we used independent component analysis (ICA) to identify signals arising from electrical reference and from volume-conducted noise based on their distributed spatial pattern across multiple electrodes and distinct power spectral features. These sources of distal electrical signals collectively accounted for 23–77% of total variance in unprocessed LFPs, as well as most of the gamma oscillation responses to the target stimulus in EEGs. Gamma oscillation power was concentrated in volume-conducted noise and was tightly coupled with the onset of licking behavior, suggesting a likely origin of muscle activity associated with body movement or orofacial movement. The removal of distal signal contamination also selectively reduced correlations of LFP/EEG signals between distant brain regions but not within the same region. Finally, the removal of contamination from distal electrical signals preserved an event-related potential (ERP) response to auditory stimuli in the frontal cortex and also increased the coupling between the frontal ERP amplitude and neuronal activity in the basal forebrain, supporting the conclusion that removing distal electrical signals unmasked local activity within LFPs. Together, these results highlight the significant contamination of LFPs by distal electrical signals and caution against the straightforward interpretation of unprocessed LFPs. Our results provide a principled approach to

Subcellular localization and logistics of integral membrane protein biogenesis in Escherichia coli.

PubMed

Bogdanov, Mikhail; Aboulwafa, Mohammad; Saier, Milton H

2013-01-01

Transporters catalyze entry and exit of molecules into and out of cells and organelles, and protein-lipid interactions influence their activities. The bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS) catalyzes transport-coupled sugar phosphorylation as well as nonvectorial sugar phosphorylation in the cytoplasm. The vectorial process is much more sensitive to the lipid environment than the nonvectorial process. Moreover, cytoplasmic micellar forms of these enzyme-porters have been identified, and non-PTS permeases have similarly been shown to exist in 'soluble' forms. The latter porters exhibit lipid-dependent activities and can adopt altered topologies by simply changing the lipid composition. Finally, intracellular membranes and vesicles exist in Escherichia coli leading to the following unanswered questions: (1) what determines whether a PTS permease catalyzes vectorial or nonvectorial sugar phosphorylation? (2) How do phospholipids influence relative amounts of the plasma membrane, intracellular membrane, inner membrane-derived vesicles and cytoplasmic micelles? (3) What regulates the route(s) of permease insertion and transfer into and between the different subcellular sites? (4) Do these various membranous forms have distinct physiological functions? (5) What methods should be utilized to study the biogenesis and interconversion of these membranous structures? While research concerning these questions is still in its infancy, answers will greatly enhance our understanding of protein-lipid interactions and how they control the activities, conformations, cellular locations and biogenesis of integral membrane proteins. Copyright © 2013 S. Karger AG, Basel.

Identification of Two Novel Endoplasmic Reticulum Body-Specific Integral Membrane Proteins1[W][OA

PubMed Central

Yamada, Kenji; Nagano, Atsushi J.; Nishina, Momoko; Hara-Nishimura, Ikuko; Nishimura, Mikio

2013-01-01

The endoplasmic reticulum (ER) body, a large compartment specific to the Brassicales, accumulates β-glucosidase and possibly plays a role in the defense against pathogens and herbivores. Although the ER body is a subdomain of the ER, it is unclear whether any ER body-specific membrane protein exists. In this study, we identified two integral membrane proteins of the ER body in Arabidopsis (Arabidopsis thaliana) and termed them MEMBRANE PROTEIN OF ENDOPLASMIC RETICULUM BODY1 (MEB1) and MEB2. In Arabidopsis, a basic helix-loop-helix transcription factor, NAI1, and an ER body component, NAI2, regulate ER body formation. The expression profiles of MEB1 and MEB2 are similar to those of NAI1, NAI2, and ER body β-glucosidase PYK10 in Arabidopsis. The expression of MEB1 and MEB2 was reduced in the nai1 mutant, indicating that NAI1 regulates the expression of MEB1 and MEB2 genes. MEB1 and MEB2 proteins localize to the ER body membrane but not to the ER network, suggesting that these proteins are specifically recruited to the ER body membrane. MEB1 and MEB2 physically interacted with ER body component NAI2, and they were diffused throughout the ER network in the nai2 mutant, which has no ER body. Heterologous expression of MEB1 and MEB2 in yeast (Saccharomyces cerevisiae) suppresses iron and manganese toxicity, suggesting that MEB1 and MEB2 are metal transporters. These results indicate that the membrane of ER bodies has specific membrane proteins and suggest that the ER body is involved in defense against metal stress as well as pathogens and herbivores. PMID:23166355

Controlled ionic condensation at the surface of a native extremophile membrane

NASA Astrophysics Data System (ADS)

Contera, Sonia Antoranz; Voïtchovsky, Kislon; Ryan, John F.

2010-02-01

At the nanoscale level biological membranes present a complex interface with the solvent. The functional dynamics and relative flexibility of membrane components together with the presence of specific ionic effects can combine to create exciting new phenomena that challenge traditional theories such as the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory or models interpreting the role of ions in terms of their ability to structure water (structure making/breaking). Here we investigate ionic effects at the surface of a highly charged extremophile membrane composed of a proton pump (bacteriorhodopsin) and archaeal lipids naturally assembled into a 2D crystal. Using amplitude-modulation atomic force microscopy (AM-AFM) in solution, we obtained sub-molecular resolution images of ion-induced surface restructuring of the membrane. We demonstrate the presence of a stiff cationic layer condensed at its extracellular surface. This layer cannot be explained by traditional continuum theories. Dynamic force spectroscopy experiments suggest that it is produced by electrostatic correlation mediated by a Manning-type condensation of ions. In contrast, the cytoplasmic surface is dominated by short-range repulsive hydration forces. These findings are relevant to archaeal bioenergetics and halophilic adaptation. Importantly, they present experimental evidence of a natural system that locally controls its interactions with the surrounding medium and challenges our current understanding of biological interfaces.At the nanoscale level biological membranes present a complex interface with the solvent. The functional dynamics and relative flexibility of membrane components together with the presence of specific ionic effects can combine to create exciting new phenomena that challenge traditional theories such as the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory or models interpreting the role of ions in terms of their ability to structure water (structure making/breaking). Here we

A membrane-anchored E-type endo-1,4-beta-glucanase is localized on Golgi and plasma membranes of higher plants.

PubMed

Brummell, D A; Catala, C; Lashbrook, C C; Bennett, A B

1997-04-29

Endo-1,4-beta-D-glucanases (EGases, EC 3.2.1.4) are enzymes produced in bacteria, fungi, and plants that hydrolyze polysaccharides possessing a 1,4-beta-D-glucan backbone. All previously identified plant EGases are E-type endoglucanases that possess signal sequences for endoplasmic reticulum entry and are secreted to the cell wall. Here we report the characterization of a novel E-type plant EGase (tomato Cel3) with a hydrophobic transmembrane domain and structure typical of type II integral membrane proteins. The predicted protein is composed of 617 amino acids and possesses seven potential sites for N-glycosylation. Cel3 mRNA accumulates in young vegetative tissues with highest abundance during periods of rapid cell expansion, but is not hormonally regulated. Antibodies raised to a recombinant Cel3 protein specifically recognized three proteins, with apparent molecular masses of 93, 88, and 53 kDa, in tomato root microsomal membranes separated by sucrose density centrifugation. The 53-kDa protein comigrated in the gradient with plasma membrane markers, the 88-kDa protein with Golgi membrane markers, and the 93-kDa protein with markers for both Golgi and plasma membranes. EGase enzyme activity was also found in regions of the density gradient corresponding to both Golgi and plasma membranes, suggesting that Cel3 EGase resides in both membrane systems, the sites of cell wall polymer biosynthesis. The in vivo function of Cel3 is not known, but the only other known membrane-anchored EGase is present in Agrobacterium tumefaciens where it is required for cellulose biosynthesis.

Multiphoton-generated localized electron plasma for membrane permeability modification in single cells

NASA Astrophysics Data System (ADS)

Merritt, T.; Leblanc, M.; McMillan, J.; Westwood, J.; Khodaparast, G. A.

2014-03-01

Successful incorporation of a specific macromolecule into a single cell would be ideal for characterizing trafficking dynamics through plasmodesmata or for studying intracellular localizations. Here, we demonstrate NIR femtosecond laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into living cells of Arabidopsis thaliana seedling stems. Based on the reactions of fluorescing vacuoles of transgenic cells and artificial cell walls comprised of nanocellulose, laser intensity and exposure time were adjusted to avoid deleterious effects. Using these plant-tailored laser parameters, cells were injected with the fluorophores and long-term dye retention was observed, all while preserving vital cell functions. This method is ideal for studies concerning cell-to-cell interactions and potentially paves the way for introducing transgenes to specific cells. This work was supported by NSF award IOS-0843372 to JHW, with additional support from and U.S. Department of Agriculture Hatch Project no. 135997, and by the Institute of Critical Technology and Applied Sciences (ICTAS) at Virginia Tech.

Self-assembled virus-membrane complexes

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

Yang, Lihua; Liang, Hongjun; Angelini, Thomas

Anionic polyelectrolytes and cationic lipid membranes can self-assemble into lamellar structures ranging from alternating layers of membranes and polyelectrolytes to 'missing layer' superlattice structures. We show that these structural differences can be understood in terms of the surface-charge-density mismatch between the polyelectrolyte and membrane components by examining complexes between cationic membranes and highly charged M13 viruses, a system that allowed us to vary the polyelectrolyte diameter independently of the charge density. Such virus-membrane complexes have pore sizes that are about ten times larger in area than DNA-membrane complexes, and can be used to package and organize large functional molecules; correlatedmore » arrays of Ru(bpy){sub 3}{sup 2+} macroionic dyes have been directly observed within the virus-membrane complexes using an electron-density reconstruction. These observations elucidate fundamental design rules for rational control of self-assembled polyelectrolyte-membrane structures, which have applications ranging from non-viral gene therapy to biomolecular templates for nanofabrication.« less

PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

PubMed Central

Su, Tizhi; Bao, Liwei; Xie, Xiujie; Lehner, Caryn L.; Cavey, Greg S.; Teknos, Theodoros N.

2013-01-01

Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA. PMID:24191200

Perspective of Membrane Technology in Dairy Industry: A Review

PubMed Central

Kumar, Pavan; Sharma, Neelesh; Ranjan, Rajeev; Kumar, Sunil; Bhat, Z. F.; Jeong, Dong Kee

2013-01-01

Membrane technology has revolutionized the dairy sector. Different types of membranes are used in the industry for various purposes like extending the shelf life of milk without exposure to heat treatment, standardization of the major components of milk for tailoring new products as well increasing yield and quality of the dairy products, and concentrating, fractionation and purification of milk components especially valuable milk proteins in their natural state. In the cheese industry, membranes increase the yield and quality of cheese and control the whey volume, by concentrating the cheese milk. With the advancement of newer technology in membrane processes, it is possible to recover growth factor from whey. With the introduction of superior quality membranes as well as newer technology, the major limitation of membranes, fouling or blockage has been overcome to a greater extent. PMID:25049918

Lipopolysaccharide Membrane Building and Simulation

PubMed Central

Jo, Sunhwan; Wu, Emilia L.; Stuhlsatz, Danielle; Klauda, Jeffery B.; Widmalm, Göran; Im, Wonpil

2015-01-01

Summary While membrane simulations are widely employed to study the structure and dynamics of various lipid bilayers and membrane proteins in the bilayers, simulations of lipopolysaccharides (LPS) in membrane environments have been limited due to its structural complexity, difficulties in building LPS-membrane systems, and lack of appropriate molecular force field. In this work, as a first step to extend CHARMM-GUI Membrane Builder to incorporate LPS molecules and to explore their structures and dynamics in membrane environments using molecular dynamics simulations, we describe step-by-step procedures to build LPS bilayer systems using CHARMM and the recently developed CHARMM carbohydrate and lipid force fields. Such procedures are illustrated by building various bilayers of Escherichia coli O6 LPS and their preliminary simulation results are given in terms of per-LPS area and density distributions of various components along the membrane normal. PMID:25753722

A single frequency component-based re-estimated MUSIC algorithm for impact localization on complex composite structures

NASA Astrophysics Data System (ADS)

Yuan, Shenfang; Bao, Qiao; Qiu, Lei; Zhong, Yongteng

2015-10-01

The growing use of composite materials on aircraft structures has attracted much attention for impact monitoring as a kind of structural health monitoring (SHM) method. Multiple signal classification (MUSIC)-based monitoring technology is a promising method because of its directional scanning ability and easy arrangement of the sensor array. However, for applications on real complex structures, some challenges still exist. The impact-induced elastic waves usually exhibit a wide-band performance, giving rise to the difficulty in obtaining the phase velocity directly. In addition, composite structures usually have obvious anisotropy, and the complex structural style of real aircrafts further enhances this performance, which greatly reduces the localization precision of the MUSIC-based method. To improve the MUSIC-based impact monitoring method, this paper first analyzes and demonstrates the influence of measurement precision of the phase velocity on the localization results of the MUSIC impact localization method. In order to improve the accuracy of the phase velocity measurement, a single frequency component extraction method is presented. Additionally, a single frequency component-based re-estimated MUSIC (SFCBR-MUSIC) algorithm is proposed to reduce the localization error caused by the anisotropy of the complex composite structure. The proposed method is verified on a real composite aircraft wing box, which has T-stiffeners and screw holes. Three typical categories of 41 impacts are monitored. Experimental results show that the SFCBR-MUSIC algorithm can localize impact on complex composite structures with an obviously improved accuracy.

An optimized ensemble local mean decomposition method for fault detection of mechanical components

NASA Astrophysics Data System (ADS)

Zhang, Chao; Li, Zhixiong; Hu, Chao; Chen, Shuai; Wang, Jianguo; Zhang, Xiaogang

2017-03-01

Mechanical transmission systems have been widely adopted in most of industrial applications, and issues related to the maintenance of these systems have attracted considerable attention in the past few decades. The recently developed ensemble local mean decomposition (ELMD) method shows satisfactory performance in fault detection of mechanical components for preventing catastrophic failures and reducing maintenance costs. However, the performance of ELMD often heavily depends on proper selection of its model parameters. To this end, this paper proposes an optimized ensemble local mean decomposition (OELMD) method to determinate an optimum set of ELMD parameters for vibration signal analysis. In OELMD, an error index termed the relative root-mean-square error (Relative RMSE) is used to evaluate the decomposition performance of ELMD with a certain amplitude of the added white noise. Once a maximum Relative RMSE, corresponding to an optimal noise amplitude, is determined, OELMD then identifies optimal noise bandwidth and ensemble number based on the Relative RMSE and signal-to-noise ratio (SNR), respectively. Thus, all three critical parameters of ELMD (i.e. noise amplitude and bandwidth, and ensemble number) are optimized by OELMD. The effectiveness of OELMD was evaluated using experimental vibration signals measured from three different mechanical components (i.e. the rolling bearing, gear and diesel engine) under faulty operation conditions.

STED Imaging of Golgi Dynamics with Cer-SiR: A Two-Component, Photostable, High-Density Lipid Probe for Live Cells.

PubMed

Erdmann, Roman S; Toomre, Derek; Schepartz, Alanna

2017-01-01

Long time-lapse super-resolution imaging in live cells requires a labeling strategy that combines a bright, photostable fluorophore with a high-density localization probe. Lipids are ideal high-density localization probes, as they are >100 times more abundant than most membrane-bound proteins and simultaneously demark the boundaries of cellular organelles. Here, we describe Cer-SiR, a two-component, high-density lipid probe that is exceptionally photostable. Cer-SiR is generated in cells via a bioorthogonal reaction of two components: a ceramide lipid tagged with trans-cyclooctene (Cer-TCO) and a reactive, photostable Si-rhodamine dye (SiR-Tz). These components assemble within the Golgi apparatus of live cells to form Cer-SiR. Cer-SiR is benign to cellular function, localizes within the Golgi at a high density, and is sufficiently photostable to enable visualization of Golgi structure and dynamics by 3D confocal or long time-lapse STED microscopy.

Independent component analysis of EEG dipole source localization in resting and action state of brain

NASA Astrophysics Data System (ADS)

Almurshedi, Ahmed; Ismail, Abd Khamim

2015-04-01

EEG source localization was studied in order to determine the location of the brain sources that are responsible for the measured potentials at the scalp electrodes using EEGLAB with Independent Component Analysis (ICA) algorithm. Neuron source locations are responsible in generating current dipoles in different states of brain through the measured potentials. The current dipole sources localization are measured by fitting an equivalent current dipole model using a non-linear optimization technique with the implementation of standardized boundary element head model. To fit dipole models to ICA components in an EEGLAB dataset, ICA decomposition is performed and appropriate components to be fitted are selected. The topographical scalp distributions of delta, theta, alpha, and beta power spectrum and cross coherence of EEG signals are observed. In close eyes condition it shows that during resting and action states of brain, alpha band was activated from occipital (O1, O2) and partial (P3, P4) area. Therefore, parieto-occipital area of brain are active in both resting and action state of brain. However cross coherence tells that there is more coherence between right and left hemisphere in action state of brain than that in the resting state. The preliminary result indicates that these potentials arise from the same generators in the brain.

The Role of Progesterone and a Novel Progesterone Receptor, Progesterone Receptor Membrane Component 1, in the Inflammatory Response of Fetal Membranes to Ureaplasma parvum Infection.

PubMed

Feng, Liping; Ransom, Carla E; Nazzal, Matthew K; Allen, Terrence K; Li, Yi-Ju; Truong, Tracy; Potts, Lauren C; Seed, Patrick C; Murtha, Amy P

2016-01-01

Ureaplasma parvum (U. parvum) is gaining recognition as an important pathogen for chorioamnionitis and preterm premature rupture of membranes. We aimed to investigate the roles of progesterone (P4) and a novel progesterone receptor, progesterone receptor membrane component 1 (PGRMC1), in the response of fetal membranes to U. parvum. Fetal membrane cells (amnion, chorion and decidua) were isolated and confirmed to be free of Mycoplasmataceae. Cells were treated with U. parvum (5x106 CFU), and adherence was quantified by qPCR. Amnion and chorion cells were transfected with scrambled siRNA or validated PGRMC1 siRNA for 72h. Cells were then treated with U. parvum for 4h with or without pretreatment with P4 (10-7 M) or ethanol for 1h. Interleukin-8 (IL-8), matrix metalloproteinase 9 (MMP9) and cyclooxygenase (COX-2) mRNA expression were quantified by qRT-PCR. Culture medium was harvested and analyzed for IL-8 and prostaglandin (PGE2) secretion by ELISA and MMP9 activity by zymography. U. parvum had a mean adherence of 15.0±0.6%, 16.9± 3.7% and 4.7±0.3% in cultured amnion, chorion and decidua cells, respectively. Exposure to U. parvum elicited significant inflammatory responses including induction of IL-8, COX-2, PGE2 and MMP9. A possible role of PGRMC1 was identified in the inhibition of U. parvum-stimulated COX-2 and MMP9 mRNA expression in chorion cells and MMP9 activity in amnion cells. On the other hand, it might enhance the U. parvum-stimulated IL-8 protein secretion in amnion cells. P4, mediated through PGRMC1, significantly inhibited U. Parvum-induced MMP9 mRNA and COX-2 mRNA expression in chorion cells. P4 appeared to attenuate U. parvum induced IL-8 mRNA expression in chorion cells, but this P4 effect might not mediated through PGRMC1. In summary, U. parvum preferentially adheres to and induces inflammatory responses in chorion and amnion cells. P4 and PGRMC1 appear to differentially modulate the inflammatory responses induced by U. parvum among amnion and

The Fluid-Mosaic Model of Membrane Structure: still relevant to understanding the structure, function and dynamics of biological membranes after more than 40 years.

PubMed

Nicolson, Garth L

2014-06-01

In 1972 the Fluid-Mosaic Membrane Model of membrane structure was proposed based on thermodynamic principals of organization of membrane lipids and proteins and available evidence of asymmetry and lateral mobility within the membrane matrix [S. J. Singer and G. L. Nicolson, Science 175 (1972) 720-731]. After over 40years, this basic model of the cell membrane remains relevant for describing the basic nano-structures of a variety of intracellular and cellular membranes of plant and animal cells and lower forms of life. In the intervening years, however, new information has documented the importance and roles of specialized membrane domains, such as lipid rafts and protein/glycoprotein complexes, in describing the macrostructure, dynamics and functions of cellular membranes as well as the roles of membrane-associated cytoskeletal fences and extracellular matrix structures in limiting the lateral diffusion and range of motion of membrane components. These newer data build on the foundation of the original model and add new layers of complexity and hierarchy, but the concepts described in the original model are still applicable today. In updated versions of the model more emphasis has been placed on the mosaic nature of the macrostructure of cellular membranes where many protein and lipid components are limited in their rotational and lateral motilities in the membrane plane, especially in their natural states where lipid-lipid, protein-protein and lipid-protein interactions as well as cell-matrix, cell-cell and intracellular membrane-associated protein and cytoskeletal interactions are important in restraining the lateral motility and range of motion of particular membrane components. The formation of specialized membrane domains and the presence of tightly packed integral membrane protein complexes due to membrane-associated fences, fenceposts and other structures are considered very important in describing membrane dynamics and architecture. These structures along

Mitochondrial and Plasma Membrane Pools of Stomatin-Like Protein 2 Coalesce at the Immunological Synapse during T Cell Activation

PubMed Central

Christie, Darah A.; Kirchhof, Mark G.; Vardhana, Santosh; Dustin, Michael L.; Madrenas, Joaquín

2012-01-01

Stomatin-like protein 2 (SLP-2) is a member of the stomatin – prohibitin – flotillin – HflC/K (SPFH) superfamily. Recent evidence indicates that SLP-2 is involved in the organization of cardiolipin-enriched microdomains in mitochondrial membranes and the regulation of mitochondrial biogenesis and function. In T cells, this role translates into enhanced T cell activation. Although the major pool of SLP-2 is associated with mitochondria, we show here that there is an additional pool of SLP-2 associated with the plasma membrane of T cells. Both plasma membrane-associated and mitochondria-associated pools of SLP-2 coalesce at the immunological synapse (IS) upon T cell activation. SLP-2 is not required for formation of IS nor for the re-localization of mitochondria to the IS because SLP-2-deficient T cells showed normal re-localization of these organelles in response to T cell activation. Interestingly, upon T cell activation, we found the surface pool of SLP-2 mostly excluded from the central supramolecular activation complex, and enriched in the peripheral area of the IS where signalling TCR microclusters are located. Based on these results, we propose that SLP-2 facilitates the compartmentalization not only of mitochondrial membranes but also of the plasma membrane into functional microdomains. In this latter location, SLP-2 may facilitate the optimal assembly of TCR signalosome components. Our data also suggest that there may be a net exchange of membrane material between mitochondria and plasma membrane, explaining the presence of some mitochondrial proteins in the plasma membrane. PMID:22623988

Stability and dynamics of membrane-spanning DNA nanopores

NASA Astrophysics Data System (ADS)

Maingi, Vishal; Burns, Jonathan R.; Uusitalo, Jaakko J.; Howorka, Stefan; Marrink, Siewert J.; Sansom, Mark S. P.

2017-03-01

Recently developed DNA-based analogues of membrane proteins have advanced synthetic biology. A fundamental question is how hydrophilic nanostructures reside in the hydrophobic environment of the membrane. Here, we use multiscale molecular dynamics (MD) simulations to explore the structure, stability and dynamics of an archetypical DNA nanotube inserted via a ring of membrane anchors into a phospholipid bilayer. Coarse-grained MD reveals that the lipids reorganize locally to interact closely with the membrane-spanning section of the DNA tube. Steered simulations along the bilayer normal establish the metastable nature of the inserted pore, yielding a force profile with barriers for membrane exit due to the membrane anchors. Atomistic, equilibrium simulations at two salt concentrations confirm the close packing of lipid around of the stably inserted DNA pore and its cation selectivity, while revealing localized structural fluctuations. The wide-ranging and detailed insight informs the design of next-generation DNA pores for synthetic biology or biomedicine.

Data-Based Locally Directed Evaluation of Vocational Education Programs. Component 3. Assessing Student Career Interests.

ERIC Educational Resources Information Center

Florida State Univ., Tallahassee. Program of Vocational Education.

Part of a system by which local education agency (LEA) personnel may evaluate secondary and postsecondary vocational education programs, this third of eight components focuses on assessment of student career interests. Procedures covered include determination of the student population to be surveyed, selection of the instrument, and discrepancy…

Mechanics of Lipid Bilayer Membranes

NASA Astrophysics Data System (ADS)

Powers, Thomas R.

All cells have membranes. The plasma membrane encapsulates the cell's interior, acting as a barrier against the outside world. In cells with nuclei (eukaryotic cells), membranes also form internal compartments (organelles) which carry out specialized tasks, such as protein modification and sorting in the case of the Golgi apparatus, and ATP production in the case of mitochondria. The main components of membranes are lipids and proteins. The proteins can be channels, carriers, receptors, catalysts, signaling molecules, or structural elements, and typically contribute a substantial fraction of the total membrane dry weight. The equilibrium properties of pure lipid membranes are relatively well-understood, and will be the main focus of this article. The framework of elasticity theory and statistical mechanics that we will develop will serve as the foundation for understanding biological phenomena such as the nonequilibrium behavior of membranes laden with ion pumps, the role of membrane elasticity in ion channel gating, and the dynamics of vesicle fission and fusion. Understanding the mechanics of lipid membranes is also important for drug encapsulation and delivery.

Effects of clary sage oil and its main components, linalool and linalyl acetate, on the plasma membrane of Candida albicans: an in vivo EPR study.

PubMed

Blaskó, Ágnes; Gazdag, Zoltán; Gróf, Pál; Máté, Gábor; Sárosi, Szilvia; Krisch, Judit; Vágvölgyi, Csaba; Makszin, Lilla; Pesti, Miklós

2017-02-01

The effects of clary sage (Salvia sclarea L.) oil (CS-oil), and its two main components, linalool (Lol) and linalyl acetate (LA), on cells of the eukaryotic human pathogen yeast Candida albicans were studied. Dynamic and thermodynamic properties of the plasma membrane were investigated by electron paramagnetic resonance (EPR) spectroscopy, with 5-doxylstearic acid (5-SASL) and 16-SASL as spin labels. The monitoring of the head group regions with 5-SASL revealed break-point frequency decrease in a temperature dependent manner of the plasma membrane between 9.55 and 13.15 °C in untreated, in CS-oil-, Lol- and LA-treated membranes. The results suggest a significant increase in fluidity of the treated plasma membranes close to the head groups. Comparison of the results observed with the two spin labels demonstrated that CS-oil and LA induced an increased level of fluidization at both depths of the plasma membrane. Whereas Lol treatment induced a less (1 %) ordered bilayer organization in the superficial regions and an increased (10 %) order of the membrane leaflet in deeper layers. Acute toxicity tests and EPR results indicated that both the apoptotic and the effects exerted on the plasma membrane fluidity depended on the composition and chemical structure of the examined materials. In comparison with the control, treatment with CS-oil, Lol or LA induced 13.0, 12.3 and 26.4 % loss respectively, of the metabolites absorbing at 260 nm, as a biological consequence of the plasma membrane fluidizing effects. Our results confirmed that clary sage oil causes plasma membrane perturbations which leads to cell apoptosis process.

Aptamer-recognized carbohydrates on the cell membrane revealed by super-resolution microscopy.

PubMed

Jing, Yingying; Cai, Mingjun; Xu, Haijiao; Zhou, Lulu; Yan, Qiuyan; Gao, Jing; Wang, Hongda

2018-04-26

Carbohydrates are one of the most important components on the cell membrane, which participate in various physiological activities, and their aberrant expression is a consequence of pathological changes. In previous studies, carbohydrate analysis basically relied on lectins. However, discrimination between lectins still exists due to their multivalent character. Furthermore, the structures obtained by carbohydrate-lectin crosslinking confuse our direct observation to some extent. Fortunately, the emergence of aptamers, which are smaller and more flexible, has provided us an unprecedented choice. Herein, an aptamer recognition method with high precise localization was developed for imaging membrane-bound N-acetylgalactosamine (GalNAc). By using direct stochastic optical reconstruction microscopy (dSTORM), we compared this aptamer recognition method with the lectin recognition method for visualizing the detailed structure of GalNAc at the nanometer scale. The results indicated that GalNAc forms irregular clusters on the cell membrane with a resolution of 23 ± 7 nm by aptamer recognition. Additionally, when treated with N-acetylgalactosidase, the aptamer-recognized GalNAc shows a more significant decrease in cluster size and localization density, thus verifying better specificity of aptamers than lectins. Collectively, our study suggests that aptamers can act as perfect substitutes for lectins in carbohydrate labeling, which will be of great potential value in the field of super-resolution fluorescence imaging.

Rapidly transported organelles containing membrane and cytoskeletal components: their relation to axonal growth

PubMed Central

1987-01-01

We have examined the movements, composition, and cellular origin of phase-dense varicosities in cultures of chick sympathetic and sensory neurons. These organelles are variable in diameter (typically between 0.2 and 2 microns) and undergo saltatory movements both towards and away from the neuronal cell body. Their mean velocities vary inversely with the size of the organelle and are greater in the retrograde than the anterograde direction. Organelles stain with the lipophilic dye 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine and with antibodies to cytoskeletal components. In cultures double-stained with antibodies to alpha-tubulin and 70-kD neurofilament protein (NF-L), approximately 40% of the organelles stain for tubulin, 30% stain for NF- L, 10% stain for both tubulin and NF-L, and 40% show no staining with either antibody. The association of cytoskeletal proteins with the organelles shows that these proteins are able to move by a form of rapid axonal transport. Under most culture conditions the predominant direction of movement is towards the cell body, suggesting that the organelles are produced at or near the growth cone. Retrograde movements continue in culture medium lacking protein or high molecular mass components and increase under conditions in which the advance of the growth cone is arrested. There is a fourfold increase in the number of organelles moving retrogradely in neurites that encounter a substratum-associated barrier to elongation; retrograde movements increase similarly in cultures exposed to cytochalasin at levels known to block growth cone advance. No previously described organelle shows behavior coordinated with axonal growth in this way. We propose that the organelles contain membrane and cytoskeletal components that have been delivered to the growth cone, by slow or fast anterograde transport, in excess of the amounts required to synthesize more axon. In view of their rapid mobility and variable contents, we suggest that they

Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering*

PubMed Central

Heinrich, Frank; Nanda, Hirsh; Goh, Haw Zan; Bachert, Collin; Lösche, Mathias; Linstedt, Adam D.

2014-01-01

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni2+-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering. PMID:24505136

Gas Separation Using Organic-Vapor-Resistent Membranes In Conjunctin With Organic-Vapor-Selective Membranes

DOEpatents

Baker, Richard W.; Pinnau, Ingo; He, Zhenjie; Da Costa, Andre R.; Daniels, Ramin; Amo, Karl D.; Wijmans, Johannes G.

2003-06-03

A process for treating a gas mixture containing at least an organic compound gas or vapor and a second gas, such as natural gas, refinery off-gas or air. The process uses two sequential membrane separation steps, one using membrane selective for the organic compound over the second gas, the other selective for the second gas over the organic vapor. The second-gas-selective membranes use a selective layer made from a polymer having repeating units of a fluorinated polymer, and demonstrate good resistance to plasticization by the organic components in the gas mixture under treatment, and good recovery after exposure to liquid aromatic hydrocarbons. The membrane steps can be combined in either order.

ATP-binding cassette transporters and sterol O-acyltransferases interact at membrane microdomains to modulate sterol uptake and esterification.

PubMed

Gulati, Sonia; Balderes, Dina; Kim, Christine; Guo, Zhongmin A; Wilcox, Lisa; Area-Gomez, Estela; Snider, Jamie; Wolinski, Heimo; Stagljar, Igor; Granato, Juliana T; Ruggles, Kelly V; DeGiorgis, Joseph A; Kohlwein, Sepp D; Schon, Eric A; Sturley, Stephen L

2015-11-01

A key component of eukaryotic lipid homeostasis is the esterification of sterols with fatty acids by sterol O-acyltransferases (SOATs). The esterification reactions are allosterically activated by their sterol substrates, the majority of which accumulate at the plasma membrane. We demonstrate that in yeast, sterol transport from the plasma membrane to the site of esterification is associated with the physical interaction of the major SOAT, acyl-coenzyme A:cholesterol acyltransferase (ACAT)-related enzyme (Are)2p, with 2 plasma membrane ATP-binding cassette (ABC) transporters: Aus1p and Pdr11p. Are2p, Aus1p, and Pdr11p, unlike the minor acyltransferase, Are1p, colocalize to sterol and sphingolipid-enriched, detergent-resistant microdomains (DRMs). Deletion of either ABC transporter results in Are2p relocalization to detergent-soluble membrane domains and a significant decrease (53-36%) in esterification of exogenous sterol. Similarly, in murine tissues, the SOAT1/Acat1 enzyme and activity localize to DRMs. This subcellular localization is diminished upon deletion of murine ABC transporters, such as Abcg1, which itself is DRM associated. We propose that the close proximity of sterol esterification and transport proteins to each other combined with their residence in lipid-enriched membrane microdomains facilitates rapid, high-capacity sterol transport and esterification, obviating any requirement for soluble intermediary proteins. © FASEB.

Association of gamma-secretase with lipid rafts in post-Golgi and endosome membranes.

PubMed

Vetrivel, Kulandaivelu S; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C; Xu, Huaxi; Thinakaran, Gopal

2004-10-22

Alzheimer's disease-associated beta-amyloid peptides (Abeta) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by beta- and gamma-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major beta-secretase in neurons is a palmitoylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the gamma-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1(-/-)/PS2(-/-) and NCT(-/-) fibroblasts, gamma-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires gamma-secretase complex assembly. Biochemical evidence shows that subunits of the gamma-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of gamma-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP.

Preliminary comparison of different immune and production components in local and imported Saanen goats reared under a sub-tropical environment.

PubMed

Barbour, Elie K; Itani, Houssam H; Sleiman, Fawwak T; Saade, Maya F; Harakeh, Steve; Nour, Afif M Abdel; Shaib, Houssam A

2012-01-01

Three objectives were included in this research work. The first objective compared different immune components in healthy mature males, mature females, and female kids of local and imported Saanen goats, reared under a sub-tropical environment. The significantly differing immune components were the blood monocyte percent, blood CD8 count, and the total white blood cell count. The second objective compared the performance of Saanen versus local does. The means of the milk yield and prolificacy of the imported Saanen does were significantly higher than those of the local does (p<0.05). The third objective compared the immune responses (hemagglutination-HA titers) and complement fixation (CF) titers in mature does of the two breeds to chicken red blood cells (c-RBC). The HA titers showed a significant seroconversion only in imported Saanen (p<0.05) but not in local does; however, the CF titers increased significantly at 4 weeks following priming with c-RBC in local (p<0.05) but not in the imported Saanen does. The impact of the differences in blood immune components and responses to antigens in the compared goats on protection potential against prevalent diseases in the sub-tropical zone of the eastern Mediterranean countries is discussed.

Lateral Membrane Heterogeneity Regulates Viral-Induced Membrane Fusion during HIV Entry.

PubMed

Molotkovsky, Rodion J; Alexandrova, Veronika V; Galimzyanov, Timur R; Jiménez-Munguía, Irene; Pavlov, Konstantin V; Batishchev, Oleg V; Akimov, Sergey A

2018-05-16

Sphingomyelin- and cholesterol- enriched membrane domains, commonly referred to as "rafts" play a crucial role in a large number of intra- and intercellular processes. Recent experiments suggest that not only the volumetric inhomogeneity of lipid distribution in rafts, but also the arrangement of the 1D boundary between the raft and the surrounding membrane is important for the membrane-associated processes. The reason is that the boundary preferentially recruits different peptides, such as HIV (human immunodeficiency virus) fusion peptide. In the present work, we report a theoretical investigation of mechanisms of influence of the raft boundary arrangement upon virus-induced membrane fusion. We theoretically predict that the raft boundary can act as an attractor for viral fusion peptides, which preferentially distribute into the vicinity of the boundary, playing the role of 'line active components' of the membrane ('linactants'). We have calculated the height of the fusion energy barrier and demonstrated that, in the case of fusion between HIV membrane and the target cell, presence of the raft boundary in the vicinity of the fusion site facilitates fusion. The results we obtained can be further generalized to be applicable to other enveloped viruses.

Two component-three dimensional catalysis

DOEpatents

Schwartz, Michael; White, James H.; Sammells, Anthony F.

2002-01-01

This invention relates to catalytic reactor membranes having a gas-impermeable membrane for transport of oxygen anions. The membrane has an oxidation surface and a reduction surface. The membrane is coated on its oxidation surface with an adherent catalyst layer and is optionally coated on its reduction surface with a catalyst that promotes reduction of an oxygen-containing species (e.g., O.sub.2, NO.sub.2, SO.sub.2, etc.) to generate oxygen anions on the membrane. The reactor has an oxidation zone and a reduction zone separated by the membrane. A component of an oxygen containing gas in the reduction zone is reduced at the membrane and a reduced species in a reactant gas in the oxidation zone of the reactor is oxidized. The reactor optionally contains a three-dimensional catalyst in the oxidation zone. The adherent catalyst layer and the three-dimensional catalyst are selected to promote a desired oxidation reaction, particularly a partial oxidation of a hydrocarbon.

Cluster Formation of Anchored Proteins Induced by Membrane-Mediated Interaction

PubMed Central

Li, Shuangyang; Zhang, Xianren; Wang, Wenchuan

2010-01-01

Abstract Computer simulations were used to study the cluster formation of anchored proteins in a membrane. The rate and extent of clustering was found to be dependent upon the hydrophobic length of the anchored proteins embedded in the membrane. The cluster formation mechanism of anchored proteins in our work was ascribed to the different local perturbations on the upper and lower monolayers of the membrane and the intermonolayer coupling. Simulation results demonstrated that only when the penetration depth of anchored proteins was larger than half the membrane thickness, could the structure of the lower monolayer be significantly deformed. Additionally, studies on the local structures of membranes indicated weak perturbation of bilayer thickness for a shallowly inserted protein, while there was significant perturbation for a more deeply inserted protein. The origin of membrane-mediated protein-protein interaction is therefore due to the local perturbation of the membrane thickness, and the entropy loss—both of which are caused by the conformation restriction on the lipid chains and the enhanced intermonolayer coupling for a deeply inserted protein. Finally, in this study we addressed the difference of cluster formation mechanisms between anchored proteins and transmembrane proteins. PMID:20513399

Patterning and lifetime of plasma membrane-localized cellulose synthase is dependent on actin organization in Arabidopsis interphase cells.

PubMed

Sampathkumar, Arun; Gutierrez, Ryan; McFarlane, Heather E; Bringmann, Martin; Lindeboom, Jelmer; Emons, Anne-Mie; Samuels, Lacey; Ketelaar, Tijs; Ehrhardt, David W; Persson, Staffan

2013-06-01

The actin and microtubule cytoskeletons regulate cell shape across phyla, from bacteria to metazoans. In organisms with cell walls, the wall acts as a primary constraint of shape, and generation of specific cell shape depends on cytoskeletal organization for wall deposition and/or cell expansion. In higher plants, cortical microtubules help to organize cell wall construction by positioning the delivery of cellulose synthase (CesA) complexes and guiding their trajectories to orient newly synthesized cellulose microfibrils. The actin cytoskeleton is required for normal distribution of CesAs to the plasma membrane, but more specific roles for actin in cell wall assembly and organization remain largely elusive. We show that the actin cytoskeleton functions to regulate the CesA delivery rate to, and lifetime of CesAs at, the plasma membrane, which affects cellulose production. Furthermore, quantitative image analyses revealed that actin organization affects CesA tracking behavior at the plasma membrane and that small CesA compartments were associated with the actin cytoskeleton. By contrast, localized insertion of CesAs adjacent to cortical microtubules was not affected by the actin organization. Hence, both actin and microtubule cytoskeletons play important roles in regulating CesA trafficking, cellulose deposition, and organization of cell wall biogenesis.

Time-delayed feedback control of breathing localized structures in a three-component reaction-diffusion system.

PubMed

Gurevich, Svetlana V

2014-10-28

The dynamics of a single breathing localized structure in a three-component reaction-diffusion system subjected to time-delayed feedback is investigated. It is shown that variation of the delay time and the feedback strength can lead either to stabilization of the breathing or to delay-induced periodic or quasi-periodic oscillations of the localized structure. A bifurcation analysis of the system in question is provided and an order parameter equation is derived that describes the dynamics of the localized structure in the vicinity of the Andronov-Hopf bifurcation. With the aid of this equation, the boundaries of the stabilization domains as well as the dependence of the oscillation radius on delay parameters can be explicitly derived, providing a robust mechanism to control the behaviour of the breathing localized structure in a straightforward manner. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Membrane Localization of Human Equilibrative Nucleoside Transporter 1 in Tumor Cells May Predict Response to Adjuvant Gemcitabine in Resected Cholangiocarcinoma Patients

PubMed Central

Deserti, Marzia; Vasuri, Francesco; Farioli, Andrea; Degiovanni, Alessio; Palloni, Andrea; Frega, Giorgio; Barbera, Maria A.; de Lorenzo, Stefania; Garajova, Ingrid; Di Marco, Mariacristina; Pinna, Antonio D.; Cescon, Matteo; Cucchetti, Alessandro; Ercolani, Giorgio; D’Errico-Grigioni, Antonietta; Pantaleo, Maria A.; Biasco, Guido; Tavolari, Simona

2016-01-01

Background. The use of gemcitabine as an adjuvant modality for cholangiocarcinoma (CC) is increasing, but limited data are available on predictive biomarkers of response. Human equilibrative nucleoside transporter 1 (hENT-1) is the major transporter involved in gemcitabine intracellular uptake. This study investigated the putative predictive role of hENT-1 localization in tumor cells of CC patients undergoing treatment with adjuvant gemcitabine. Methods. Seventy-one consecutive patients with resected CC receiving adjuvant gemcitabine at our center were retrospectively analyzed by immunohistochemistry for hENT-1 localization in tumor cells. The main outcome measure was disease-free survival (DFS). Hazard ratios (HRs) of relapse and associated 95% confidence intervals (CIs) were obtained from proportional hazards regression models stratified on quintiles of propensity score. Results. Twenty-three (32.4%) cases were negative for hENT-1, 22 (31.0%) were positive in the cytoplasm only, and 26 (36.6%) showed concomitant cytoplasm/membrane staining. Patients with membrane hENT-1 had a longer DFS (HR 0.49, 95% CI 0.24–0.99, p = .046) than those who were negative or positive only in the cytoplasm of tumor cells. Notably, the association between DFS and membrane hENT-1 was dependent on the number of gemcitabine cycles (one to two cycles: HR 0.96, 95% CI 0.34–2.68; three to four cycles: HR 0.99, 95% CI 0.34–2.90; five to six cycles: HR 0.27, 95% CI 0.10–0.77). Conclusion. hENT-1 localization on tumor cell membrane may predict response to adjuvant gemcitabine in CC patients receiving more than four cycles of chemotherapy. Further prospective randomized trials on larger populations are required to confirm these preliminary results, so that optimal gemcitabine-based chemotherapy may be tailored for CC patients in the adjuvant setting. Implications for Practice: Gemcitabine is becoming an increasingly used adjuvant modality in cholangiocarcinoma (CC), but limited data are

Membrane Localization of Human Equilibrative Nucleoside Transporter 1 in Tumor Cells May Predict Response to Adjuvant Gemcitabine in Resected Cholangiocarcinoma Patients.

PubMed

Brandi, Giovanni; Deserti, Marzia; Vasuri, Francesco; Farioli, Andrea; Degiovanni, Alessio; Palloni, Andrea; Frega, Giorgio; Barbera, Maria A; de Lorenzo, Stefania; Garajova, Ingrid; Di Marco, Mariacristina; Pinna, Antonio D; Cescon, Matteo; Cucchetti, Alessandro; Ercolani, Giorgio; D'Errico-Grigioni, Antonietta; Pantaleo, Maria A; Biasco, Guido; Tavolari, Simona

2016-05-01

The use of gemcitabine as an adjuvant modality for cholangiocarcinoma (CC) is increasing, but limited data are available on predictive biomarkers of response. Human equilibrative nucleoside transporter 1 (hENT-1) is the major transporter involved in gemcitabine intracellular uptake. This study investigated the putative predictive role of hENT-1 localization in tumor cells of CC patients undergoing treatment with adjuvant gemcitabine. Seventy-one consecutive patients with resected CC receiving adjuvant gemcitabine at our center were retrospectively analyzed by immunohistochemistry for hENT-1 localization in tumor cells. The main outcome measure was disease-free survival (DFS). Hazard ratios (HRs) of relapse and associated 95% confidence intervals (CIs) were obtained from proportional hazards regression models stratified on quintiles of propensity score. Twenty-three (32.4%) cases were negative for hENT-1, 22 (31.0%) were positive in the cytoplasm only, and 26 (36.6%) showed concomitant cytoplasm/membrane staining. Patients with membrane hENT-1 had a longer DFS (HR 0.49, 95% CI 0.24-0.99, p = .046) than those who were negative or positive only in the cytoplasm of tumor cells. Notably, the association between DFS and membrane hENT-1 was dependent on the number of gemcitabine cycles (one to two cycles: HR 0.96, 95% CI 0.34-2.68; three to four cycles: HR 0.99, 95% CI 0.34-2.90; five to six cycles: HR 0.27, 95% CI 0.10-0.77). hENT-1 localization on tumor cell membrane may predict response to adjuvant gemcitabine in CC patients receiving more than four cycles of chemotherapy. Further prospective randomized trials on larger populations are required to confirm these preliminary results, so that optimal gemcitabine-based chemotherapy may be tailored for CC patients in the adjuvant setting. Gemcitabine is becoming an increasingly used adjuvant modality in cholangiocarcinoma (CC), but limited data are available on predictive biomarkers of response. In this study, patients

Adducin forms a bridge between the erythrocyte membrane and its cytoskeleton and regulates membrane cohesion

PubMed Central

Anong, William A.; Franco, Taina; Chu, Haiyan; Weis, Tahlia L.; Devlin, Emily E.; Bodine, David M.; An, Xiuli; Mohandas, Narla

2009-01-01

The erythrocyte membrane skeleton is the best understood cytoskeleton. Because its protein components have homologs in virtually all other cells, the membrane serves as a fundamental model of biologic membranes. Modern textbooks portray the membrane as a 2-dimensional spectrin-based membrane skeleton attached to a lipid bilayer through 2 linkages: band 3–ankyrin–β-spectrin and glycophorin C–protein 4.1–β-spectrin.1–7 Although evidence supports an essential role for the first bridge in regulating membrane cohesion, rupture of the glycophorin C–protein 4.1 interaction has little effect on membrane stability.8 We demonstrate the existence of a novel band 3–adducin–spectrin bridge that connects the spectrin/actin/protein 4.1 junctional complex to the bilayer. As rupture of this bridge leads to spontaneous membrane fragmentation, we conclude that the band 3–adducin–spectrin bridge is important to membrane stability. The required relocation of part of the band 3 population to the spectrin/actin junctional complex and its formation of a new bridge with adducin necessitates a significant revision of accepted models of the erythrocyte membrane. PMID:19567882

Membrane-associated proteomics of chickpea identifies Sad1/UNC-84 protein (CaSUN1), a novel component of dehydration signaling

NASA Astrophysics Data System (ADS)

Jaiswal, Dinesh Kumar; Mishra, Poonam; Subba, Pratigya; Rathi, Divya; Chakraborty, Subhra; Chakraborty, Niranjan

2014-02-01

Dehydration affects almost all the physiological processes including those that result in the accumulation of misfolded proteins in the endoplasmic reticulum (ER), which in turn elicits a highly conserved signaling, the unfolded protein response (UPR). We investigated the dehydration-responsive membrane-associated proteome of a legume, chickpea, by 2-DE coupled with mass spectrometry. A total of 184 protein spots were significantly altered over a dehydration treatment of 120 h. Among the differentially expressed proteins, a non-canonical SUN domain protein, designated CaSUN1 (Cicer arietinum Sad1/UNC-84), was identified. CaSUN1 localized to the nuclear membrane and ER, besides small vacuolar vesicles. The transcripts were downregulated by both abiotic and biotic stresses, but not by abscisic acid treatment. Overexpression of CaSUN1 conferred stress tolerance in transgenic Arabidopsis. Furthermore, functional complementation of the yeast mutant, slp1, could rescue its growth defects. We propose that the function of CaSUN1 in stress response might be regulated via UPR signaling.

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

PubMed

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

2017-05-04

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

Three-dimensional localization and optical imaging of objects in turbid media with independent component analysis.

PubMed

Xu, M; Alrubaiee, M; Gayen, S K; Alfano, R R

2005-04-01

A new approach for optical imaging and localization of objects in turbid media that makes use of the independent component analysis (ICA) from information theory is demonstrated. Experimental arrangement realizes a multisource illumination of a turbid medium with embedded objects and a multidetector acquisition of transmitted light on the medium boundary. The resulting spatial diversity and multiple angular observations provide robust data for three-dimensional localization and characterization of absorbing and scattering inhomogeneities embedded in a turbid medium. ICA of the perturbations in the spatial intensity distribution on the medium boundary sorts out the embedded objects, and their locations are obtained from Green's function analysis based on any appropriate light propagation model. Imaging experiments were carried out on two highly scattering samples of thickness approximately 50 times the transport mean-free path of the respective medium. One turbid medium had two embedded absorptive objects, and the other had four scattering objects. An independent component separation of the signal, in conjunction with diffusive photon migration theory, was used to locate the embedded inhomogeneities. In both cases, improved lateral and axial localizations of the objects over the result obtained by use of common photon migration reconstruction algorithms were achieved. The approach is applicable to different medium geometries, can be used with any suitable photon propagation model, and is amenable to near-real-time imaging applications.

Pepper pathogenesis-related protein 4c is a plasma membrane-localized cysteine protease inhibitor that is required for plant cell death and defense signaling.

PubMed

Kim, Nak Hyun; Hwang, Byung Kook

2015-01-01

Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

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

Glucuronylated core 1 glycans are required for precise localization of neuromuscular junctions and normal formation of basement membranes on Drosophila muscles.

PubMed

Itoh, Kazuyoshi; Akimoto, Yoshihiro; Kondo, Shu; Ichimiya, Tomomi; Aoki, Kazuhiro; Tiemeyer, Michael; Nishihara, Shoko

2018-04-15

T antigen (Galβ1-3GalNAcα1-Ser/Thr) is an evolutionary-conserved mucin-type core 1 glycan structure in animals synthesized by core 1 β1,3-galactosyltransferase 1 (C1GalT1). Previous studies showed that T antigen produced by Drosophila C1GalT1 (dC1GalT1) was expressed in various tissues and dC1GalT1 loss in larvae led to various defects, including decreased number of circulating hemocytes, hyper-differentiation of hematopoietic stem cells in lymph glands, malformation of the central nervous system, mislocalization of neuromuscular junction (NMJ) boutons, and ultrastructural abnormalities in NMJs and muscle cells. Although glucuronylated T antigen (GlcAβ1-3Galβ1-3GalNAcα1-Ser/Thr) has been identified in Drosophila, the physiological function of this structure has not yet been clarified. In this study, for the first time, we unraveled biological roles of glucuronylated T antigen. Our data show that in Drosophila, glucuronylation of T antigen is predominantly carried out by Drosophila β1,3-glucuronyltransferase-P (dGlcAT-P). We created dGlcAT-P null mutants and found that mutant larvae showed lower expression of glucuronylated T antigen on the muscles and at NMJs. Furthermore, mislocalization of NMJ boutons and a partial loss of the basement membrane components collagen IV (Col IV) and nidogen (Ndg) at the muscle 6/7 boundary were observed. Those two phenotypes were correlated and identical to previously described phenotypes in dC1GalT1 mutant larvae. In addition, dGlcAT-P null mutants exhibited fewer NMJ branches on muscles 6/7. Moreover, ultrastructural analysis revealed that basement membranes that lacked Col IV and Ndg were significantly deformed. We also found that the loss of dGlcAT-P expression caused ultrastructural defects in NMJ boutons. Finally, we showed a genetic interaction between dGlcAT-P and dC1GalT1. Therefore, these results demonstrate that glucuronylated core 1 glycans synthesized by dGlcAT-P are key modulators of NMJ bouton localization

[Germ cell membrane lipids in spermatogenesis].

PubMed

Wang, Ting; Shi, Xiao; Quan, Song

2016-05-01

Spermatogenesis is a complex developmental process in which a diploid progenitor germ cell transforms into highly specialized spermatozoa. During spermatogenesis, membrane remodeling takes place, and cell membrane permeability and liquidity undergo phase-specific changes, which are all associated with the alteration of membrane lipids. Lipids are important components of the germ cell membrane, whose volume and ratio fluctuate in different phases of spermatogenesis. Abnormal lipid metabolism can cause spermatogenic dysfunction and consequently male infertility. Germ cell membrane lipids are mainly composed of cholesterol, phospholipids and glycolipids, which play critical roles in cell adhesion and signal transduction during spermatogenesis. An insight into the correlation of membrane lipids with spermatogenesis helps us to better understand the mechanisms of spermatogenesis and provide new approaches to the diagnosis and treatment of male infertility.

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization.

PubMed

Roux, Kyle J; Crisp, Melissa L; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L; Burke, Brian

2009-02-17

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin-Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1-3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning.

C60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

NASA Astrophysics Data System (ADS)

Russ, K. A.; Elvati, P.; Parsonage, T. L.; Dews, A.; Jarvis, J. A.; Ray, M.; Schneider, B.; Smith, P. J. S.; Williamson, P. T. F.; Violi, A.; Philbert, M. A.

2016-02-01

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. The evidence suggests marginal uptake of C60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Computational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal deformation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. The surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern

Trafficking-deficient long QT syndrome mutation KCNQ1-T587M confers severe clinical phenotype by impairment of KCNH2 membrane localization: evidence for clinically significant IKr-IKs alpha-subunit interaction.

PubMed

Biliczki, Peter; Girmatsion, Zenawit; Brandes, Ralf P; Harenkamp, Sabine; Pitard, Bruno; Charpentier, Flavien; Hébert, Terence E; Hohnloser, Stefan H; Baró, Isabelle; Nattel, Stanley; Ehrlich, Joachim R

2009-12-01

KCNQ1-T587M is a trafficking-deficient long QT syndrome (LQTS) missense mutation. Affected patients exhibit severe clinical phenotypes that are not explained by the mutant's effects on I(Ks). Previous work showed a KCNH2 and KCNQ1 alpha-subunit interaction that increases KCNH2 membrane localization and function. We hypothesized that failure of trafficking-deficient KCNQ1-T587M to enhance KCNH2 membrane expression could reduce KCNH2 current versus wild-type KCNQ1 (KCNQ1-WT), contributing to the LQTS phenotype of KCNQ1-T587M carriers. Patch-clamp, protein biochemical studies, confocal imaging, and in vivo transfection of guinea pig cardiomyocytes were performed. KCNQ1-T587M failed to generate functional current when coexpressed with KCNE1 and caused haploinsufficiency when coexpressed with KCNQ1-WT/KCNE1. Coexpression of KCNQ1-WT with KCNH2 increased I(KCNH2) versus KCNH2 alone (P <.05). Immunoblots and confocal microscopy indicated increased plasma membrane localization of KCNH2 alpha-subunits in cells cotransfected with KCNQ1-WT plasmid, while total KCNH2 protein synthesis and KCNH2 glycosylation remained unaffected, which suggests a chaperone effect of KCNQ1-WT to enhance the membrane localization of KCNH2. KCNH2 also coimmunoprecipitated with KCNQ1-WT. Although KCNQ1-T587M coprecipitated with KCNH2, the mutant was retained intracellularly and failed to increase KCNH2 membrane localization, abolishing the KCNQ1-WT chaperone function and reducing I(KCNH2) upon coexpression substantially compared with coexpression with KCNQ1-WT (P <.05). In vivo transfection of KCNQ1-T587M in guinea pigs suppressed I(Kr) in isolated cardiomyocytes. The trafficking-deficient LQTS mutation KCNQ1-T587M fails to show the chaperoning function that enhances KCNH2 membrane localization with KCNQ1-WT. This novel mechanism results in reduced I(KCNH2), which would be expected to decrease repolarization reserve and synergize with reduced I(KCNQ1) caused directly by the mutation, potentially

Application of Novel Anion-Exchange Blend Membranes (AEBMs) to Vanadium Redox Flow Batteries.

PubMed

Cho, Hyeongrae; Krieg, Henning M; Kerres, Jochen A

2018-06-19

Both cation-exchange membranes and anion-exchange membranes are used as ion conducting membranes in vanadium redox flow batteries (VRFBs). Anion-exchange membranes (AEMs) are applied in vanadium redox flow batteries due to the high blocking property of vanadium ions via the Donnan exclusion effect. In this study, novel anion-exchange blend membranes (AEBMs) were prepared, characterized, and applied in VRFBs. Bromomethylated poly(2,6-dimethyl-1,4-phenylene oxide), poly[(1-(4,4′-diphenylether)-5-oxybenzimidazole)-benzimidazole] (PBI-OO) and sulfonated polyether sulfone polymer were combined to prepare 3-component AEBMs with 1,2,4,5-tetramethylimidazole (TMIm) for quaternization. 3-component AEBMs showed significantly enhanced chemical and mechanical properties compared with those of 2-component AEBMs, resulting in an improved performance in VRFBs. The compositions of the anion-exchange polymers in 3-component AEBMs were systematically varied to optimize the AEBMs for the redox-flow battery application. While the 3-component AEBMs showed comparable efficiencies with Nafion ® 212 membranes, they displayed improved vanadium ions cross-over as was confirmed by open circuit voltage tests and capacity fade tests conducted in VRFBs. In addition, one of the synthesized 3-component AEBM had a superior coulombic efficiency and capacity retention in a charging⁻discharging test over 300 cycles at a current density of 40 mA/cm². It can thus be concluded that 3-component AEBMs are promising candidates for long-term operation in VRFBs.

Superresolution Imaging of Human Cytomegalovirus vMIA Localization in Sub-Mitochondrial Compartments

PubMed Central

Bhuvanendran, Shivaprasad; Salka, Kyle; Rainey, Kristin; Sreetama, Sen Chandra; Williams, Elizabeth; Leeker, Margretha; Prasad, Vidhya; Boyd, Jonathan; Patterson, George H.; Jaiswal, Jyoti K.; Colberg-Poley, Anamaris M.

2014-01-01

The human cytomegalovirus (HCMV) viral mitochondria-localized inhibitor of apoptosis (vMIA) protein, traffics to mitochondria-associated membranes (MAM), where the endoplasmic reticulum (ER) contacts the outer mitochondrial membrane (OMM). vMIA association with the MAM has not been visualized by imaging. Here, we have visualized this by using a combination of confocal and superresolution imaging. Deconvolution of confocal microscopy images shows vMIA localizes away from mitochondrial matrix at the Mitochondria-ER interface. By gated stimulated emission depletion (GSTED) imaging, we show that along this interface vMIA is distributed in clusters. Through multicolor, multifocal structured illumination microscopy (MSIM), we find vMIA clusters localize away from MitoTracker Red, indicating its OMM localization. GSTED and MSIM imaging show vMIA exists in clusters of ~100–150 nm, which is consistent with the cluster size determined by Photoactivated Localization Microscopy (PALM). With these diverse superresolution approaches, we have imaged the clustered distribution of vMIA at the OMM adjacent to the ER. Our findings directly compare the relative advantages of each of these superresolution imaging modalities for imaging components of the MAM and sub-mitochondrial compartments. These studies establish the ability of superresolution imaging to provide valuable insight into viral protein location, particularly in the sub-mitochondrial compartments, and into their clustered organization. PMID:24721787

Transmembrane protein sorting driven by membrane curvature

NASA Astrophysics Data System (ADS)

Strahl, H.; Ronneau, S.; González, B. Solana; Klutsch, D.; Schaffner-Barbero, C.; Hamoen, L. W.

2015-11-01

The intricate structure of prokaryotic and eukaryotic cells depends on the ability to target proteins to specific cellular locations. In most cases, we have a poor understanding of the underlying mechanisms. A typical example is the assembly of bacterial chemoreceptors at cell poles. Here we show that the classical chemoreceptor TlpA of Bacillus subtilis does not localize according to the consensus stochastic nucleation mechanism but accumulates at strongly curved membrane areas generated during cell division. This preference was confirmed by accumulation at non-septal curved membranes. Localization appears to be an intrinsic property of the protein complex and does not rely on chemoreceptor clustering, as was previously shown for Escherichia coli. By constructing specific amino-acid substitutions, we demonstrate that the preference for strongly curved membranes arises from the curved shape of chemoreceptor trimer of dimers. These findings demonstrate that the intrinsic shape of transmembrane proteins can determine their cellular localization.

Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

PubMed Central

Mendanha, S.A.; Anjos, J.L.V.; Silva, A.H.M.; Alonso, A.

2012-01-01

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H2O2). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H2O2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H2O2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation. PMID:22473321

Lipid raft components cholesterol and sphingomyelin increase H+/OH− permeability of phosphatidylcholine membranes

PubMed Central

Gensure, Rebekah H.; Zeidel, Mark L.; Hill, Warren G.

2006-01-01

H+/OH− permeation through lipid bilayers occurs at anomalously high rates and the determinants of proton flux through membranes are poorly understood. Since all life depends on proton gradients, it is important to develop a greater understanding of proton leak phenomena. We have used stopped-flow fluorimetry to probe the influence of two lipid raft components, chol (cholesterol) and SM (sphingomyelin), on H+/OH− and water permeability. Increasing the concentrations of both lipids in POPC (palmitoyl-2-oleoyl phosphatidylcholine) liposomes decreased water permeability in a concentration-dependent manner, an effect that correlated with increased lipid order. Surprisingly, proton flux was increased by increasing the concentration of chol and SM. The chol effect was complex with molar concentrations of 17.9, 33 and 45.7% giving 2.8-fold (P<0.01), 2.2-fold (P<0.001) and 5.1-fold (P<0.001) increases in H+/OH− permeability from a baseline of 2.4×10−2 cm/s. SM at 10 mole% effected a 2.8-fold increase (P<0.01), whereas 20 and 30 mole% enhanced permeability by 3.6-fold (P<0.05) and 4.1-fold respectively (P<0.05). Supplementing membranes containing chol with SM did not enhance H+/OH− permeability. Of interest was the finding that chol addition to soya-bean lipids decreased H+/OH− permeability, consistent with an earlier report [Ira and Krishnamoorthy (2001) J. Phys. Chem. B 105, 1484–1488]. We speculate that the presence of proton carriers in crude lipid extracts might contribute to this result. We conclude that (i) chol and SM specifically and independently increase rates of proton permeation in POPC bilayers, (ii) domains enriched in these lipids or domain interfaces may represent regions with high H+/OH− conductivity, (iii) H+/OH− fluxes are not governed by lipid order and (iv) chol can inhibit or promote H+/OH− permeability depending on the total lipid environment. Theories of proton permeation are discussed in the light of these results. PMID

Standard plane localization in ultrasound by radial component model and selective search.

PubMed

Ni, Dong; Yang, Xin; Chen, Xin; Chin, Chien-Ting; Chen, Siping; Heng, Pheng Ann; Li, Shengli; Qin, Jing; Wang, Tianfu

2014-11-01

Acquisition of the standard plane is crucial for medical ultrasound diagnosis. However, this process requires substantial experience and a thorough knowledge of human anatomy. Therefore it is very challenging for novices and even time consuming for experienced examiners. We proposed a hierarchical, supervised learning framework for automatically detecting the standard plane from consecutive 2-D ultrasound images. We tested this technique by developing a system that localizes the fetal abdominal standard plane from ultrasound video by detecting three key anatomical structures: the stomach bubble, umbilical vein and spine. We first proposed a novel radial component-based model to describe the geometric constraints of these key anatomical structures. We then introduced a novel selective search method which exploits the vessel probability algorithm to produce probable locations for the spine and umbilical vein. Next, using component classifiers trained by random forests, we detected the key anatomical structures at their probable locations within the regions constrained by the radial component-based model. Finally, a second-level classifier combined the results from the component detection to identify an ultrasound image as either a "fetal abdominal standard plane" or a "non- fetal abdominal standard plane." Experimental results on 223 fetal abdomen videos showed that the detection accuracy of our method was as high as 85.6% and significantly outperformed both the full abdomen and the separate anatomy detection methods without geometric constraints. The experimental results demonstrated that our system shows great promise for application to clinical practice. Copyright © 2014 World Federation for Ultrasound in Medicine & Biology. Published by Elsevier Inc. All rights reserved.

Comparative evaluation of a bioabsorbable collagen membrane and connective tissue graft in the treatment of localized gingival recession: A clinical study

PubMed Central

Babu, Harsha Mysore; Gujjari, Sheela Kumar; Prasad, Deepak; Sehgal, Praveen Kumar; Srinivasan, Aishwarya

2011-01-01

Background: Gingival recession (GR) can result in root sensitivity, esthetic concern to the patient, and predilection to root caries. The purpose of this randomized clinical study was to evaluate (1) the effect of guided tissue regeneration (GTR) procedure using a bioabsorbable collagen membrane, in comparison to autogenous subepithelial connective tissue graft (SCTG) for root coverage in localized gingival recession defects; and (2) the change in width of keratinized gingiva following these two procedures. Materials and Methods: A total of 10 cases, showing at least two localized Miller's Class I or Class II gingival recession, participated in this study. In a split mouth design, the pairs of defects were randomly assigned for treatment with either SCTG (SCTG Group) or GTR-based collagen membrane (GTRC Group). Both the grafts were covered with coronally advanced flap. Recession depth (RD), recession width (RW), width of keratinized gingiva (KG), probing depth (PD), relative attachment level (RAL), plaque index (PI), and gingival index (GI) were recorded at baseline, 3 and 6 months postoperatively. Results: Six months following root coverage procedures, the mean root coverage was found to be 84.84% ± 16.81% and 84.0% ± 15.19% in SCTG Group and GTRC Group, respectively. The mean keratinized gingival width increase was 1.50 ± 0.70 mm and 2.30 ± 0.67 mm in the SCTG and GTRC group, respectively, which was not statistically significant. Conclusion: It may be concluded that resorbable collagen membrane can be a reliable alternative to autogenous connective tissue graft in the treatment of gingival recession. PMID:22368359

Differential solubilization of inner plasma membrane leaflet components by Lubrol WX and Triton X-100.

PubMed

Delaunay, Jean-Louis; Breton, Michelyne; Trugnan, Germain; Maurice, Michèle

2008-01-01

A commonly-used method for analysing raft membrane domains is based on their resistance to extraction by non-ionic detergents at 4 degrees C. However, the selectivity of different detergents in defining raft membrane domains has been questioned. We have compared the lipid composition of detergent-resistant membranes (DRMs) obtained after Triton X-100 or Lubrol WX extraction in MDCK cells in order to understand the differential effect of these detergents on membranes and their selectivity in solubilizing or not proteins. Both Lubrol and Triton DRMs were enriched with cholesterol over the lysate, thus exhibiting characteristics consistent with the properties of membrane rafts. However, the two DRM fractions differed considerably in the ratio between lipids of the inner and outer membrane leaflets. Lubrol DRMs were especially enriched with phosphatidylethanolamine, including polyunsaturated species with long fatty acyl chains. Lubrol and Triton DRMs also differed in the amount of raft transmembrane proteins and raft proteins anchored to the cytoplasmic leaflet. Our results suggest that the inner side of rafts is enriched with phosphatidylethanolamine and cholesterol, and is more solubilized by Triton X-100 than by Lubrol WX.

Intracellular localization of a group II chaperonin indicates a membrane-related function

NASA Technical Reports Server (NTRS)

Trent, Jonathan D.; Kagawa, Hiromi K.; Paavola, Chad D.; McMillan, R. Andrew; Howard, Jeanie; Jahnke, Linda; Lavin, Colleen; Embaye, Tsegereda; Henze, Christopher E.

2003-01-01

Chaperonins are protein complexes that are believed to function as part of a protein folding system in the cytoplasm of the cell. We observed, however, that the group II chaperonins known as rosettasomes in the hyperthermophilic archaeon Sulfolobus shibatae, are not cytoplasmic but membrane associated. This association was observed in cultures grown at 60 degrees C and 76 degrees C or heat-shocked at 85 degrees C by using immunofluorescence microscopy and in thick sections of rapidly frozen cells grown at 76 degrees C by using immunogold electron microscopy. We observed that increased abundance of rosettasomes after heat shock correlated with decreased membrane permeability at lethal temperature (92 degrees C). This change in permeability was not seen in cells heat-shocked in the presence of the amino acid analogue azetidine 2-carboxylic acid, indicating functional protein synthesis influences permeability. Azetidine experiments also indicated that observed heat-induced changes in lipid composition in S. shibatae could not account for changes in membrane permeability. Rosettasomes purified from cultures grown at 60 degrees C and 76 degrees C or heat-shocked at 85 degrees C bind to liposomes made from either the bipolar tetraether lipids of Sulfolobus or a variety of artificial lipid mixtures. The presence of rosettasomes did not significantly change the transition temperature of liposomes, as indicated by differential scanning calorimetry, or the proton permeability of liposomes, as indicated by pyranine fluorescence. We propose that these group II chaperonins function as a structural element in the natural membrane based on their intracellular location, the correlation between their functional abundance and membrane permeability, and their potential distribution on the membrane surface.

Protein Diffusion on Charged Membranes: A Dynamic Mean-Field Model Describes Time Evolution and Lipid Reorganization

PubMed Central

Khelashvili, George; Weinstein, Harel; Harries, Daniel

2008-01-01

As charged macromolecules adsorb and diffuse on cell membranes in a large variety of cell signaling processes, they can attract or repel oppositely charged lipids. This results in lateral membrane rearrangement and affects the dynamics of protein function. To address such processes quantitatively we introduce a dynamic mean-field scheme that allows self-consistent calculations of the equilibrium state of membrane-protein complexes after such lateral reorganization of the membrane components, and serves to probe kinetic details of the process. Applicable to membranes with heterogeneous compositions containing several types of lipids, this comprehensive method accounts for mobile salt ions and charged macromolecules in three dimensions, as well as for lateral demixing of charged and net-neutral lipids in the membrane plane. In our model, the mobility of membrane components is governed by the diffusion-like Cahn-Hilliard equation, while the local electrochemical potential is based on nonlinear Poisson-Boltzmann theory. We illustrate the method by applying it to the adsorption of the anionic polypeptide poly-Lysine on negatively charged lipid membranes composed of binary mixtures of neutral and monovalent lipids, or onto ternary mixtures of neutral, monovalent, and multivalent lipids. Consistent with previous calculations and experiments, our results show that at steady-state multivalent lipids (such as PIP2), but not monovalent lipid (such as phosphatidylserine), will segregate near the adsorbing macromolecules. To address the corresponding diffusion of the adsorbing protein in the membrane plane, we couple lipid mobility with the propagation of the adsorbing protein through a dynamic Monte Carlo scheme. We find that due to their higher mobility dictated by the electrochemical potential, multivalent lipids such as PIP2 more quickly segregate near oppositely charged proteins than do monovalent lipids, even though their diffusion constants may be similar. The

Ero1alpha requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM).

PubMed

Gilady, Susanna Y; Bui, Michael; Lynes, Emily M; Benson, Matthew D; Watts, Russell; Vance, Jean E; Simmen, Thomas

2010-09-01

Protein secretion from the endoplasmic reticulum (ER) requires the enzymatic activity of chaperones and oxidoreductases that fold polypeptides and form disulfide bonds within newly synthesized proteins. The best-characterized ER redox relay depends on the transfer of oxidizing equivalents from molecular oxygen through ER oxidoreductin 1 (Ero1) and protein disulfide isomerase to nascent polypeptides. The formation of disulfide bonds is, however, not the sole function of ER oxidoreductases, which are also important regulators of ER calcium homeostasis. Given the role of human Ero1alpha in the regulation of the calcium release by inositol 1,4,5-trisphosphate receptors during the onset of apoptosis, we hypothesized that Ero1alpha may have a redox-sensitive localization to specific domains of the ER. Our results show that within the ER, Ero1alpha is almost exclusively found on the mitochondria-associated membrane (MAM). The localization of Ero1alpha on the MAM is dependent on oxidizing conditions within the ER. Chemical reduction of the ER environment, but not ER stress in general leads to release of Ero1alpha from the MAM. In addition, the correct localization of Ero1alpha to the MAM also requires normoxic conditions, but not ongoing oxidative phosphorylation.

Subcellular localization of pituitary enzymes

NASA Technical Reports Server (NTRS)

Smith, R. E.

1970-01-01

A cytochemical procedure is reported for identifying subcellular sites of enzymes hydrolyzing beta-naphthylamine substrates, and to study the sites of reaction product localization in cells of various tissues. Investigations using the substrate Leu 4-methoxy-8-naphthylamine, a capture with hexonium pararosaniline, and the final chelation of osmium have identified the hydrolyzing enzyme of rat liver cells; this enzyme localized on cell membranes with intense deposition in the areas of the parcanaliculi. The study of cells in the anterior pituitary of the rat showed the deposition of reaction product on cell membrane; and on the membranes of secretion granules contained within the cell. The deposition of reaction product on the cell membrane however showed no increase or decrease with changes in the physiological state of the gland and release of secretion granules from specific cells.

Aspirin locally disrupts the liquid-ordered phase

NASA Astrophysics Data System (ADS)

Alsop, Richard J.; Himbert, Sebastian; Dhaliwal, Alexander; Schmalzl, Karin; Rheinstädter, Maikel C.

2018-02-01

Local structure and dynamics of lipid membranes play an important role in membrane function. The diffusion of small molecules, the curvature of lipids around a protein and the existence of cholesterol-rich lipid domains (rafts) are examples for the membrane to serve as a functional interface. The collective fluctuations of lipid tails, in particular, are relevant for diffusion of membrane constituents and small molecules in and across membranes, and for structure and formation of membrane domains. We studied the effect of aspirin (acetylsalicylic acid, ASA) on local structure and dynamics of membranes composed of dimyristoylphosphocholine (DMPC) and cholesterol. Aspirin is a common analgesic, but is also used in the treatment of cholesterol. Using coherent inelastic neutron scattering experiments and molecular dynamics (MD) simulations, we present evidence that ASA binds to liquid-ordered, raft-like domains and disturbs domain organization and dampens collective fluctuations. By hydrogen-bonding to lipid molecules, ASA forms `superfluid' complexes with lipid molecules that can organize laterally in superlattices and suppress cholesterol's ordering effect.

Association of γ-Secretase with Lipid Rafts in Post-Golgi and Endosome Membranes*

PubMed Central

Vetrivel, Kulandaivelu S.; Cheng, Haipeng; Lin, William; Sakurai, Takashi; Li, Tong; Nukina, Nobuyuki; Wong, Philip C.; Xu, Huaxi; Thinakaran, Gopal

2005-01-01

Alzheimer’s disease-associated β-amyloid peptides (Aβ) are generated by the sequential proteolytic processing of amyloid precursor protein (APP) by β- and γ-secretases. There is growing evidence that cholesterol- and sphingolipid-rich membrane microdomains are involved in regulating trafficking and processing of APP. BACE1, the major γ-secretase in neurons is a palmi-toylated transmembrane protein that resides in lipid rafts. A subset of APP is subject to amyloidogenic processing by BACE1 in lipid rafts, and this process depends on the integrity of lipid rafts. Here we describe the association of all four components of the γ-secretase complex, namely presenilin 1 (PS1)-derived fragments, mature nicastrin, APH-1, and PEN-2, with cholesterol-rich detergent insoluble membrane (DIM) domains of non-neuronal cells and neurons that fulfill the criteria of lipid rafts. In PS1−/−/PS2−/− and NCT−/− fibroblasts, γ-secretase components that still remain fail to become detergent-resistant, suggesting that raft association requires γ-secretase complex assembly. Biochemical evidence shows that subunits of the γ-secretase complex and three TGN/endosome-resident SNAREs cofractionate in sucrose density gradients, and show similar solubility or insolubility characteristics in distinct non-ionic and zwitterionic detergents, indicative of their co-residence in membrane microdomains with similar protein-lipid composition. This notion is confirmed using magnetic immunoisolation of PS1- or syntaxin 6-positive membrane patches from a mixture of membranes with similar buoyant densities following Lubrol WX extraction or sonication, and gradient centrifugation. These findings are consistent with the localization of γ-secretase in lipid raft microdomains of post-Golgi and endosomes, organelles previously implicated in amyloidogenic processing of APP. PMID:15322084

Raft membrane domains: from a liquid-ordered membrane phase to a site of pathogen attack.

PubMed

van der Goot, F G; Harder, T

2001-04-01

While the existence of cholesterol/sphingolipid (raft) membrane domains in the plasma membrane is now supported by strong experimental evidence, the structure of these domains, their size, their dynamics, and their molecular composition remain to be understood. Raft domains are thought to represent a specific physical state of lipid bilayers, the liquid-ordered phase. Recent observations suggest that in the mammalian plasma membrane small raft domains in ordered lipid phases are in a dynamic equilibrium with a less ordered membrane environment. Rafts may be enlarged and/or stabilized by protein-mediated cross-linking of raft-associated components. These changes of plasma membrane structure are perceived by the cells as signals, most likely an important element of immunoreceptor signalling. Pathogens abuse raft domains on the host cell plasma membrane as concentration devices, as signalling platforms and/or entry sites into the cell. Elucidation of these interactions requires a detailed understanding raft structure and dynamics. Copyright 2001 Academic Press.

Lysosomal degradation of membrane lipids.

PubMed

Kolter, Thomas; Sandhoff, Konrad

2010-05-03

The constitutive degradation of membrane components takes place in the acidic compartments of a cell, the endosomes and lysosomes. Sites of lipid degradation are intralysosomal membranes that are formed in endosomes, where the lipid composition is adjusted for degradation. Cholesterol is sorted out of the inner membranes, their content in bis(monoacylglycero)phosphate increases, and, most likely, sphingomyelin is degraded to ceramide. Together with endosomal and lysosomal lipid-binding proteins, the Niemann-Pick disease, type C2-protein, the GM2-activator, and the saposins sap-A, -B, -C, and -D, a suitable membrane lipid composition is required for degradation of complex lipids by hydrolytic enzymes. Copyright 2009 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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

PubMed

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

2015-05-01

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

Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

PubMed

Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

2014-01-01

Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

Dynamics and Size of Cross-Linking-Induced Lipid Nanodomains in Model Membranes

PubMed Central

Štefl, Martin; Šachl, Radek; Humpolíčková, Jana; Cebecauer, Marek; Macháň, Radek; Kolářová, Marie; Johansson, Lennart B.-Å.; Hof, Martin

2012-01-01

Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Förster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of ∼8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation. PMID:22824274

Reversible and irreversible low-pressure membrane foulants in drinking water treatment: Identification by principal component analysis of fluorescence EEM and mitigation by biofiltration pretreatment.

PubMed

Peldszus, Sigrid; Hallé, Cynthia; Peiris, Ramila H; Hamouda, Mohamed; Jin, Xiaohui; Legge, Raymond L; Budman, Hector; Moresoli, Christine; Huck, Peter M

2011-10-15

With the increased use of membranes in drinking water treatment, fouling--particularly the hydraulically irreversible type--remains the main operating issue that hinders performance and increases operational costs. The main challenge in assessing fouling potential of feed water is to accurately detect and quantify feed water constituents responsible for membrane fouling. Utilizing fluorescence excitation-emission matrices (EEM), protein-like substances, humic and fulvic acids, and particulate/colloidal matter can be detected with high sensitivity in surface waters. The application of principal component analysis to fluorescence EEMs allowed estimation of the impact of surface water constituents on reversible and irreversible membrane fouling. This technique was applied to experimental data from a two year bench-scale study that included thirteen experiments investigating the fouling potential of Grand River water (Ontario, Canada) and the effect of biofiltration pre-treatment on the level of foulants during ultrafiltration (UF). Results showed that, although the content of protein-like substances in this membrane feed water (=biofiltered natural water) was much lower than commonly found in wastewater applications, the content of protein-like substances was still highly correlated with irreversible fouling of the UF membrane. In addition, there is evidence that protein-like substances and particulate/colloidal matter formed a combined fouling layer, which contributed to both reversible and irreversible fouling. It is suggested that fouling transitions from a reversible to an irreversible regime depending on feed composition and operating time. Direct biofiltration without prior coagulant addition reduced the protein-like content of the membrane feed water which in turn reduced the irreversible fouling potential for UF membranes. Biofilters also decreased reversible fouling, and for both types of fouling higher biofilter contact times were beneficial. Copyright �

Proteomic analysis of symbiosome membranes in Cnidaria-dinoflagellate endosymbiosis.

PubMed

Peng, Shao-En; Wang, Yu-Bao; Wang, Li-Hsueh; Chen, Wan-Nan Uang; Lu, Chi-Yu; Fang, Lee-Shing; Chen, Chii-Shiarng

2010-03-01

Symbiosomes are specific intracellular membrane-bound vacuoles containing microalgae in a mutualistic Cnidaria (host)-dinoflagellate (symbiont) association. The symbiosome membrane is originally derived from host plasma membranes during phagocytosis of the symbiont; however, its molecular components and functions are not clear. In order to investigate the protein components of the symbiosome membranes, homogenous symbiosomes were isolated from the sea anemone Aiptasia pulchella and their purities and membrane intactness examined by Western blot analysis for host contaminants and microscopic analysis using various fluorescent probes, respectively. Pure and intact symbiosomes were then subjected to biotinylation by a cell impermeant agent (Biotin-XX sulfosuccinimidyl ester) to label membrane surface proteins. The biotinylated proteins, both Triton X-100 soluble and insoluble fractions, were subjected to 2-D SDS-PAGE and identified by MS using an LC-nano-ESI-MS/MS. A total of 17 proteins were identified. Based on their different subcellular origins and functional categories, it indicates that symbiosome membranes serve as the interface for interaction between host and symbiont by fulfilling several crucial cellular functions such as those of membrane receptors/cell recognition, cytoskeletal remodeling, ATP synthesis/proton homeostasis, transporters, stress responses/chaperones, and anti-apoptosis. The results of proteomic analysis not only indicate the molecular identity of the symbiosome membrane, but also provide insight into the possible role of symbiosome membranes during the endosymbiotic association.

Synaptic membrane rafts: traffic lights for local neurotrophin signaling?

PubMed

Zonta, Barbara; Minichiello, Liliana

2013-10-18

Lipid rafts, cholesterol and lipid rich microdomains, are believed to play important roles as platforms for the partitioning of transmembrane and synaptic proteins involved in synaptic signaling, plasticity, and maintenance. There is increasing evidence of a physical interaction between post-synaptic densities and post-synaptic lipid rafts. Localization of proteins within lipid rafts is highly regulated, and therefore lipid rafts may function as traffic lights modulating and fine-tuning neuronal signaling. The tyrosine kinase neurotrophin receptors (Trk) and the low-affinity p75 neurotrophin receptor (p75(NTR)) are enriched in neuronal lipid rafts together with the intermediates of downstream signaling pathways, suggesting a possible role of rafts in neurotrophin signaling. Moreover, neurotrophins and their receptors are involved in the regulation of cholesterol metabolism. Cholesterol is an important component of lipid rafts and its depletion leads to gradual loss of synapses, underscoring the importance of lipid rafts for proper neuronal function. Here, we review and discuss the idea that translocation of neurotrophin receptors in synaptic rafts may account for the selectivity of their transduced signals.

Localization and Function of the Membrane-bound Riboflavin in the Na+-translocating NADH:Quinone Oxidoreductase (Na+-NQR) from Vibrio cholerae*

PubMed Central

Casutt, Marco S.; Huber, Tamara; Brunisholz, René; Tao, Minli; Fritz, Günter; Steuber, Julia

2010-01-01

The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the human pathogen Vibrio cholerae is a respiratory membrane protein complex that couples the oxidation of NADH to the transport of Na+ across the bacterial membrane. The Na+-NQR comprises the six subunits NqrABCDEF, but the stoichiometry and arrangement of these subunits are unknown. Redox-active cofactors are FAD and a 2Fe-2S cluster on NqrF, covalently attached FMNs on NqrB and NqrC, and riboflavin and ubiquinone-8 with unknown localization in the complex. By analyzing the cofactor content and NADH oxidation activity of subcomplexes of the Na+-NQR lacking individual subunits, the riboflavin cofactor was unequivocally assigned to the membrane-bound NqrB subunit. Quantitative analysis of the N-terminal amino acids of the holo-complex revealed that NqrB is present in a single copy in the holo-complex. It is concluded that the hydrophobic NqrB harbors one riboflavin in addition to its covalently attached FMN. The catalytic role of two flavins in subunit NqrB during the reduction of ubiquinone to ubiquinol by the Na+-NQR is discussed. PMID:20558724

Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.

PubMed

Vosolsobě, Stanislav; Petrášek, Jan; Schwarzerová, Kateřina

2017-05-01

The plant-specific DREPP protein family comprises proteins that were shown to regulate the actin and microtubular cytoskeleton in a calcium-dependent manner. Our phylogenetic analysis showed that DREPPs first appeared in ferns and that DREPPs have a rapid and plastic evolutionary history in plants. Arabidopsis DREPP paralogues called AtMDP25/PCaP1 and AtMAP18/PCaP2 are N-myristoylated, which has been reported as a key factor in plasma membrane localization. Here we show that N-myristoylation is neither conserved nor ancestral for the DREPP family. Instead, by using confocal microscopy and a new method for quantitative evaluation of protein membrane localization, we show that DREPPs rely on two mechanisms ensuring their plasma membrane localization. These include N-myristoylation and electrostatic interaction of a polybasic amino acid cluster. We propose that various plasma membrane association mechanisms resulting from the evolutionary plasticity of DREPPs are important for refining plasma membrane interaction of these signalling proteins under various conditions and in various cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Lipid composition affects the rate of photosensitized dissipation of cross-membrane diffusion potential on liposomes

PubMed Central

Ytzhak, Shany; Wuskell, Joseph P.; Loew, Leslie M.; Ehrenberg, Benjamin

2010-01-01

Hydrophobic or amphiphilic tetrapyrrole sensitizers are taken up by cells and are usually located in cellular lipid membranes. Singlet oxygen is photogenerated by the sensitizer and it diffuses in the membrane and causes oxidative damage to membrane components. This damage can occur to membrane lipids and to membrane-localized proteins. Depolarization of the Nernst electric potential on cells’ membranes has been observed in cellular photosensitization, but it was not established whether lipid oxidation is a relevant factor leading to abolishing the resting potential of cells’ membranes and to their death. In this work we studied the effect of liposomes’ lipid composition on the kinetics of hematoporphyrin-photosensitized dissipation of K+-diffusion electric potential that was generated across the membranes. We employed an electrochromic voltage-sensitive spectroscopic probe that possesses a high fluorescence signal response to the potential. We found a correlation between the structure and unsaturation of lipids and the leakage of the membrane, following photosensitization. As the extent of non-conjugated unsaturation of the lipids is increased from 1 to 6 double bonds, the kinetics of depolarization become faster. We also found that the kinetics of depolarization is affected by the percentage of the unsaturated lipids in the liposome: as the fraction of the unsaturated lipids increases the leakage trough the membrane is enhanced. When liposomes are composed of a lipid mixture similar to that of natural membranes and photosensitization is being carried out under usual photodynamic therapy (PDT) conditions, photodamage to the lipids is not likely to cause enhanced permeability of ions through the membrane, which would have been a mechanism that leads to cell death. PMID:20536150

Separation of the global and local components in functional near-infrared spectroscopy signals using principal component spatial filtering

PubMed Central

Zhang, Xian; Noah, Jack Adam; Hirsch, Joy

2016-01-01

Abstract. Global systemic effects not specific to a task can be prominent in functional near-infrared spectroscopy (fNIRS) signals and the separation of task-specific fNIRS signals and global nonspecific effects is challenging due to waveform correlations. We describe a principal component spatial filter algorithm for separation of the global and local effects. The effectiveness of the approach is demonstrated using fNIRS signals acquired during a right finger-thumb tapping task where the response patterns are well established. Both the temporal waveforms and the spatial pattern consistencies between oxyhemoglobin and deoxyhemoglobin signals are significantly improved, consistent with the basic physiological basis of fNIRS signals and the expected pattern of activity associated with the task. PMID:26866047

Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis.

PubMed

Takeda, Tetsuya; Robinson, Iain M; Savoian, Matthew M; Griffiths, John R; Whetton, Anthony D; McMahon, Harvey T; Glover, David M

2013-08-07

Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.

Association of p60c-src with endosomal membranes in mammalian fibroblasts

PubMed Central

1992-01-01

We have examined the subcellular localization of p60c-src in mammalian fibroblasts. Analysis of indirect immunofluorescence by three- dimensional optical sectioning microscopy revealed a granular cytoplasmic staining that co-localized with the microtubule organizing center. Immunofluorescence experiments with antibodies against a number of membrane markers demonstrated a striking co-localization between p60c-src and the cation-dependent mannose-6-phosphate receptor (CI- MPR), a marker that identifies endosomes. Both p60c-src and the CI-MPR were found to cluster at the spindle poles throughout mitosis. In addition, treatment of interphase and mitotic cells with brefeldin A resulted in a clustering of p60c-src and CI-MPR at a peri-centriolar position. Biochemical fractionation of cellular membranes showed that a major proportion of p60c-src co-enriched with endocytic membranes. Treatment of membranes containing HRP to alter their apparent density also altered the density of p60c-src-containing membranes. Similar density shift experiments with total cellular membranes revealed that the majority of membrane-associated p60c-src in the cell is associated with endosomes, while very little is associated with plasma membranes. These results support a role for p60c-src in the regulation of endosomal membranes and protein trafficking. PMID:1378446

Preparation of thermo-responsive membranes. II.

PubMed

Nozawa, I; Suzuki, Y; Sato, S; Sugibayashi, K; Morimoto, Y

1991-05-01

Two types of liquid crystal (LC)-immobilized membranes were prepared by a soaking method and sandwich method to control the permeation of indomethacin, as a model drug, in response to local and systemic fever. Monooxyethylene trimethylolpropane tristearate (MTTS) was used as a model LC because it has a gel-liquid crystal phase transition temperature near the body temperature, 39-40 degrees C in phosphate buffered saline (pH 7.4). Two porous polypropylene (PP) membranes were soaked into 20% MTTS chloroform solution in the soaking method, and two PP membranes were poured with the melted MTTS and pressed in the sandwich method. Thermo-response efficacy of the soaked membrane was dependent upon the content of MTTS in MTTS membrane, and the MTTS content above the void volume of PP membrane (38%) was needed for high efficacy. On the other hand, the sandwich membrane exhibited higher thermo-response efficacy than the soaked membrane, because more LC was embedded in the pores of sandwich membrane than that of the soaked membrane. The sandwich membrane permeation of indomethacin was sharply controlled by temperature changes between 32 and 38 degrees C.

Localized Ras signaling at the leading edge regulates PI3K, cell polarity, and directional cell movement

PubMed Central

Sasaki, Atsuo T.; Chun, Cheryl; Takeda, Kosuke; Firtel, Richard A.

2004-01-01

During chemotaxis, receptors and heterotrimeric G-protein subunits are distributed and activated almost uniformly along the cell membrane, whereas PI(3,4,5)P3, the product of phosphatidylinositol 3-kinase (PI3K), accumulates locally at the leading edge. The key intermediate event that creates this strong PI(3,4,5)P3 asymmetry remains unclear. Here, we show that Ras is rapidly and transiently activated in response to chemoattractant stimulation and regulates PI3K activity. Ras activation occurs at the leading edge of chemotaxing cells, and this local activation is independent of the F-actin cytoskeleton, whereas PI3K localization is dependent on F-actin polymerization. Inhibition of Ras results in severe defects in directional movement, indicating that Ras is an upstream component of the cell's compass. These results support a mechanism by which localized Ras activation mediates leading edge formation through activation of basal PI3K present on the plasma membrane and other Ras effectors required for chemotaxis. A feedback loop, mediated through localized F-actin polymerization, recruits cytosolic PI3K to the leading edge to amplify the signal. PMID:15534002

Bacillus subtilis actin-like protein MreB influences the positioning of the replication machinery and requires membrane proteins MreC/D and other actin-like proteins for proper localization.

PubMed

Defeu Soufo, Hervé Joël; Graumann, Peter L

2005-03-03

Bacterial actin-like proteins have been shown to perform essential functions in several aspects of cellular physiology. They affect cell growth, cell shape, chromosome segregation and polar localization of proteins, and localize as helical filaments underneath the cell membrane. Bacillus subtilis MreB and Mbl have been shown to perform dynamic motor like movements within cells, extending along helical tracks in a time scale of few seconds. In this work, we show that Bacillus subtilis MreB has a dual role, both in the formation of rod cell shape, and in chromosome segregation, however, its function in cell shape is distinct from that of MreC. Additionally, MreB is important for the localization of the replication machinery to the cell centre, which becomes aberrant soon after depletion of MreB. 3D image reconstructions suggest that frequently, MreB filaments consist of several discontinuous helical filaments with varying length. The localization of MreB was abnormal in cells with decondensed chromosomes, as well as during depletion of Mbl, MreBH and of the MreC/MreD proteins, which we show localize to the cell membrane. Thus, proper positioning of MreB filaments depends on and is affected by a variety of factors in the cell. Our data provide genetic and cytological links between MreB and the membrane, as well as with other actin like proteins, and further supports the connection of MreB with the chromosome. The functional dependence on MreB of the localization of the replication machinery suggests that the replisome is not anchored at the cell centre, but is positioned in a dynamic manner.

Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus.

PubMed

Pragman, Alexa A; Yarwood, Jeremy M; Tripp, Timothy J; Schlievert, Patrick M

2004-04-01

Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.

Cytochemical localization of Na+, K+-ATPase in the rat hepatocyte.

PubMed Central

Blitzer, B L; Boyer, J L

1978-01-01

The enzyme Na+,5+-ATPase was cytochemically localized in the rat hepatocyte by a modification of the Ernst potassium-dependent nitrophenyl phosphatase technique. Measurement of nitrophenol release from 50-micrometer liver slices confirmed the presence of ouabain-inhibitable nitrophenyl phosphatase activity that increased over the 30-min incubation period. Electron micrographs demonstrated that sinusoidal and lateral membrane reaction product deposition was K+-dependent, Mg++-dependent, inhibited by ouabain but not by alkaline phosphatase inhibitors, and was localized to the cytoplasmic side of the membrane. In contrast, canalicular reaction product was K+-independent, Mg++-dependent, inhibited by alkaline phosphatase inhibitors but not by ouabain, and was localized to the luminal side of the membrane. These findings indicate that Na+,K+-ATPase is localized to the sinusoidal and lateral portions of the rat hepatocyte plasma membrane and is not detectable on the bile canaliculus where alkaline phosphatase is confined. This basolateral localization of Na+,K+-ATPase is similar to that found in epithelia where secretion is also directed across the apical membrane. Images PMID:213446

Low molecular weight components in an aquatic humic substance as characterized by membrane dialysis and orbitrap mass spectrometry.

PubMed

Remucal, Christina K; Cory, Rose M; Sander, Michael; McNeill, Kristopher

2012-09-04

Suwannee River fulvic acid (SRFA) was dialyzed through a 100-500 molecular weight cutoff dialysis membrane, and the dialysate and retentate were analyzed by UV-visible absorption and high-resolution Orbitrap mass spectrometry (MS). A significant fraction (36% based on dissolved organic carbon) of SRFA passed through the dialysis membrane. The fraction of SRFA in the dialysate had a different UV-visible absorption spectrum and was enriched in low molecular weight molecules with a more aliphatic composition relative to the initial SRFA solution. Comparison of the SRFA spectra collected by Orbitrap MS and Fourier transform ion cyclotron resonance MS (FT-ICR MS) demonstrated that the mass accuracy of the Orbitrap MS is sufficient for determination of unique molecular formulas of compounds with masses <600 Da in a complex mixture, such as SRFA. The most intense masses detected by Orbitrap MS were found in the 100-200 Da mass range. Many of these low molecular masses corresponded to molecular formulas of previously identified compounds in organic matter, lignin, and plants, and the use of the standard addition method provided an upper concentration estimate of selected target compounds in SRFA. Collectively, these results provide evidence that SRFA contains low molecular weight components that are present individually or in loosely bound assemblies.

Regulation of T-cell receptor signalling by membrane microdomains

PubMed Central

Razzaq, Tahir M; Ozegbe, Patricia; Jury, Elizabeth C; Sembi, Phupinder; Blackwell, Nathan M; Kabouridis, Panagiotis S

2004-01-01

There is now considerable evidence suggesting that the plasma membrane of mammalian cells is compartmentalized by functional lipid raft microdomains. These structures are assemblies of specialized lipids and proteins and have been implicated in diverse biological functions. Analysis of their protein content using proteomics and other methods revealed enrichment of signalling proteins, suggesting a role for these domains in intracellular signalling. In T lymphocytes, structure/function experiments and complementary pharmacological studies have shown that raft microdomains control the localization and function of proteins which are components of signalling pathways regulated by the T-cell antigen receptor (TCR). Based on these studies, a model for TCR phosphorylation in lipid rafts is presented. However, despite substantial progress in the field, critical questions remain. For example, it is unclear if membrane rafts represent a homogeneous population and if their structure is modified upon TCR stimulation. In the future, proteomics and the parallel development of complementary analytical methods will undoubtedly contribute in further delineating the role of lipid rafts in signal transduction mechanisms. PMID:15554919

C 60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

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

Russ, K. A.; Elvati, P.; Parsonage, T. L.

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C 60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. Themore » evidence suggests marginal uptake of C 60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Compu-tational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal defor-mation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. Lastly, the surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.« less

C 60 fullerene localization and membrane interactions in RAW 264.7 immortalized mouse macrophages

DOE PAGES

Russ, K. A.; Elvati, P.; Parsonage, T. L.; ...

2016-01-01

There continues to be a significant increase in the number and complexity of hydrophobic nanomaterials that are engineered for a variety of commercial purposes making human exposure a significant health concern. This study uses a combination of biophysical, biochemical and computational methods to probe potential mechanisms for uptake of C 60 nanoparticles into various compartments of living immune cells. Cultures of RAW 264.7 immortalized murine macrophage were used as a canonical model of immune-competent cells that are likely to provide the first line of defense following inhalation. Modes of entry studied were endocytosis/pinocytosis and passive permeation of cellular membranes. Themore » evidence suggests marginal uptake of C 60 clusters is achieved through endocytosis/pinocytosis, and that passive diffusion into membranes provides a significant source of biologically-available nanomaterial. Compu-tational modeling of both a single molecule and a small cluster of fullerenes predicts that low concentrations of fullerenes enter the membrane individually and produce limited perturbation; however, at higher concentrations the clusters in the membrane causes deformation of the membrane. These findings are bolstered by nuclear magnetic resonance (NMR) of model membranes that reveal defor-mation of the cell membrane upon exposure to high concentrations of fullerenes. The atomistic and NMR models fail to explain escape of the particle out of biological membranes, but are limited to idealized systems that do not completely recapitulate the complexity of cell membranes. Lastly, the surprising contribution of passive modes of cellular entry provides new avenues for toxicological research that go beyond the pharmacological inhibition of bulk transport systems such as pinocytosis.« less

Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

PubMed Central

Sciacca, Michele F.M.; Kotler, Samuel A.; Brender, Jeffrey R.; Chen, Jennifer; Lee, Dong-kuk; Ramamoorthy, Ayyalusamy

2012-01-01

Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ1–40, defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ1–40 is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer’s disease. PMID:22947931

Membrane materials for storing biological samples intended for comparative nanotoxicological testing

NASA Astrophysics Data System (ADS)

Metelkin, A.; Kuznetsov, D.; Kolesnikov, E.; Chuprunov, K.; Kondakov, S.; Osipov, A.; Samsonova, J.

2015-11-01

The study is aimed at identifying the samples of most promising membrane materials for storing dry specimens of biological fluids (Dried Blood Spots, DBS technology). Existing sampling systems using cellulose fiber filter paper have a number of drawbacks such as uneven distribution of the sample spot, dependence of the spot spreading area on the individual biosample properties, incomplete washing-off of the sample due to partially inconvertible sorption of blood components on cellulose fibers, etc. Samples of membrane materials based on cellulose, polymers and glass fiber with applied biosamples were studied using methods of scanning electron microscopy, FT-IR spectroscopy and surface-wetting measurement. It was discovered that cellulose-based membrane materials sorb components of biological fluids inside their structure, while membranes based on glass fiber display almost no interaction with the samples and biological fluid components dry to films in the membrane pores between the structural fibers. This characteristic, together with the fact that membrane materials based on glass fiber possess sufficient strength, high wetting properties and good storage capacity, attests them as promising material for dry samples of biological fluids storage systems.

Micron-scale plasma membrane curvature is recognized by the septin cytoskeleton

PubMed Central

Bridges, Andrew A.; Jentzsch, Maximilian S.; Oakes, Patrick W.; Occhipinti, Patricia

2016-01-01

Cells change shape in response to diverse environmental and developmental conditions, creating topologies with micron-scale features. Although individual proteins can sense nanometer-scale membrane curvature, it is unclear if a cell could also use nanometer-scale components to sense micron-scale contours, such as the cytokinetic furrow and base of neuronal branches. Septins are filament-forming proteins that serve as signaling platforms and are frequently associated with areas of the plasma membrane where there is micron-scale curvature, including the cytokinetic furrow and the base of cell protrusions. We report here that fungal and human septins are able to distinguish between different degrees of micron-scale curvature in cells. By preparing supported lipid bilayers on beads of different curvature, we reconstitute and measure the intrinsic septin curvature preference. We conclude that micron-scale curvature recognition is a fundamental property of the septin cytoskeleton that provides the cell with a mechanism to know its local shape. PMID:27044896

Tracking inorganic foulants irreversibly accumulated on low-pressure membranes for treating surface water.

PubMed

Yamamura, Hiroshi; Kimura, Katsuki; Higuchi, Kumiko; Watanabe, Yoshimasa; Ding, Qing; Hafuka, Akira

2015-12-15

While low-pressure membrane filtration processes (i.e., microfiltration and ultrafiltration) can offer precise filtration than sand filtration, they pose the problem of reduced efficiency due to membrane fouling. Although many studies have examined membrane fouling by organic substances, there is still not enough data available concerning membrane fouling by inorganic substances. The present research investigated changes in the amounts of inorganic components deposited on the surface of membrane filters over time using membrane specimens sampled thirteen times at arbitrary time intervals during pilot testing in order to determine the mechanism by which irreversible fouling by inorganic substances progresses. The experiments showed that the inorganic components that primarily contribute to irreversible fouling vary as filtration continues. It was discovered that, in the initial stage of operation, the main membrane-fouling substance was iron, whereas the primary membrane-fouling substances when operation finished were manganese, calcium, and silica. The amount of iron accumulated on the membrane increased up to the thirtieth day of operation, after which it reached a steady state. After the accumulation of iron became static, subsequent accumulation of manganese was observed. The fact that the removal rates of these inorganic components also increased gradually shows that the size of the exclusion pores of the membrane filter narrows as operation continues. Studying particle size distributions of inorganic components contained in source water revealed that while many iron particles are approximately the same size as membrane pores, the fraction of manganese particles slightly smaller than the pores in diameter was large. From these results, it is surmised that iron particles approximately the same size as the pores block them soon after the start of operation, and as the membrane pores narrow with the development of fouling, they become further blocked by manganese

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport.

PubMed

Szczerba, Mark W; Britto, Dev T; Balkos, Konstantine D; Kronzucker, Herbert J

2008-01-01

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.

Characterization of the Leptospiral Outer Membrane and Description of Three Novel Leptospiral Membrane Proteins

PubMed Central

Haake, David A.; Matsunaga, James

2002-01-01

The outer membrane (OM) of the mammalian pathogen Leptospira kirschneri was isolated in the form of membrane vesicles by alkaline plasmolysis and separated from the protoplasmic cylinder by sucrose density gradient ultracentrifugation. All four components of the alkaline plasmolysis buffer, including 1.0 M NaCl, 27% sucrose (wt/vol), 2 mM EDTA, and 10 mM Tris (pH 9), were required for efficient OM release, as judged by recovery of leptospiral lipopolysaccharide. Two populations of OM vesicles (OMVs) were recovered, with peak concentrations found in the sucrose gradient at densities of 1.16 and 1.18 g/ml. Transmission electron microscopy revealed that the more buoyant OMV population was smaller (<0.1 μm in diameter) than the denser OMV population (0.2 to 0.3 μm in diameter). The densities of both populations of OMVs were distinct from that of the protoplasmic-cylinder material, which was found in the sucrose gradient at a density of 1.20 g/ml. The OMV fractions were free of protoplasmic-cylinder material, as judged by immunoblotting with antibodies to the endoflagellar sheath protein, heat shock protein GroEL, and two novel cytoplasmic membrane proteins, lipoprotein LipL31 and transmembrane protein ImpL63. The protein components of the OMVs were characterized by one- and two-dimensional immunoblotting and found to include previously described OM proteins (OMPs), including the porin OmpL1; the lipoproteins LipL32, LipL36, and LipL41; and the peripheral membrane protein P31LipL45. A number of less well-characterized OMPs were also identified, including those with molecular masses of 16, 21, 21.5, 22, 31, 36, 44, 48, 90, and 116 kDa. The 48-kDa OMP was identified as a novel OM lipoprotein designated LipL48. The use of membrane-specific markers in OM isolation techniques facilitates an accurate description of the leptospiral OM and its components. PMID:12183539

Interaction of Defensins with Model Cell Membranes

NASA Astrophysics Data System (ADS)

Sanders, Lori K.; Schmidt, Nathan W.; Yang, Lihua; Mishra, Abhijit; Gordon, Vernita D.; Selsted, Michael E.; Wong, Gerard C. L.

2009-03-01

Antimicrobial peptides (AMPs) comprise a key component of innate immunity for a wide range of multicellular organisms. For many AMPs, activity comes from their ability to selectively disrupt and lyse bacterial cell membranes. There are a number of proposed models for this action, but the detailed molecular mechanism of selective membrane permeation remains unclear. Theta defensins are circularized peptides with a high degree of selectivity. We investigate the interaction of model bacterial and eukaryotic cell membranes with theta defensins RTD-1, BTD-7, and compare them to protegrin PG-1, a prototypical AMP, using synchrotron small angle x-ray scattering (SAXS). The relationship between membrane composition and peptide induced changes in membrane curvature and topology is examined. By comparing the membrane phase behavior induced by these different peptides we will discuss the importance of amino acid composition and placement on membrane rearrangement.

Sudden death of entanglement and non-locality in two- and three-component quantum systems

NASA Astrophysics Data System (ADS)

Ann, Kevin

2011-12-01

Quantum entanglement and non-locality are non-classical characteristics of quantum states with phase coherence that are of central importance to physics, and relevant to the foundations of quantum mechanics and quantum information science. This thesis examines quantum entanglement and non-locality in two- and three-component quantum states with phase coherence when they are subject to statistically independent, classical, Markovian, phase noise in various combinations at the local and collective level. Because this noise reduces phase coherence, it can also reduce quantum entanglement and Bell non-locality. After introducing and contextualizing the research, the results are presented in three broad areas. The first area characterizes the relative time scales of decoherence and disentanglement in 2 x 2 and 3 x 3 quantum states, as well as the various subsystems of the two classes of entangled tripartite two-level quantum states. In all cases, it was found that disentanglement time scales are less than or equal to decoherence time scales. The second area examines the finite-time loss of entanglement, even as quantum state coherence is lost only asymptotically in time due to local dephasing noise, a phenomenon entitled "Entanglement Sudden Death" (ESD). Extending the initial discovery in the simplest 2 x 2 case, ESD is shown to exist in all other systems where mixed-state entanglement measures exist, the 2 x 3 and d x d systems, for finite d > 2. The third area concerns non-locality, which is a physical phenomenon independent of quantum mechanics and related to, though fundamentally different from, entanglement. Non-locality, as quantified by classes of Bell inequalities, is shown to be lost in finite time, even when decoherence occurs only asymptotically. This phenomenon was named "Bell Non-locality Sudden Death" (BNSD).

Plasma membrane microorganization of LR73 multidrug-resistant cells revealed by FCS

NASA Astrophysics Data System (ADS)

Winckler, Pascale; Jaffiol, Rodolphe; Cailler, Aurélie; Morjani, Hamid; Jeannesson, Pierre; Deturche, Régis

2011-03-01

Tumoral cells could present a multidrug resistance (MDR) to chemotherapeutic treatments. This drug resistance would be associated to biomechanisms occurring at the plasma membrane level, involving modification of membrane fluidity, drug permeability, presence of microdomains (rafts, caveolae...), and membrane proteins overexpression such as Pglycoprotein. Fluorescence correlation spectroscopy (FCS) is the relevant method to investigate locally the fluidity of biological membranes through the lateral diffusion of a fluorescent membrane probe. Thus, we use FCS to monitor the plasma membrane local organization of LR73 carcinoma cells and three derived multidrug-resistant cancer cells lines. Measurements were conducted at the single cell level, which enabled us to get a detailed overview of the plasma membrane microviscosity distribution of each cell line studied. Moreover, we propose 2D diffusion simulation based on a Monte Carlo model to investigate the membrane organisation in terms of microdomains. This simulation allows us to relate the differences in the fluidity distributions with microorganization changes in plasma membrane of MDR cells.

The Us2 gene product of herpes simplex virus 2 is a membrane-associated ubiquitin-interacting protein.

PubMed

Kang, Ming-Hsi; Roy, Bibhuti B; Finnen, Renée L; Le Sage, Valerie; Johnston, Susan M; Zhang, Hui; Banfield, Bruce W

2013-09-01

The Us2 gene encodes a tegument protein that is conserved in most members of the Alphaherpesvirinae. Previous studies on the pseudorabies virus (PRV) Us2 ortholog indicated that it is prenylated, associates with membranes, and spatially regulates the enzymatic activity of the MAP (mitogen-activated protein) kinase ERK (extracellular signal-related kinase) through direct binding and sequestration of ERK at the cytoplasmic face of the plasma membrane. Here we present an analysis of the herpes simplex virus 2 (HSV-2) Us2 ortholog and demonstrate that, like PRV Us2, HSV-2 Us2 is a virion component and that, unlike PRV Us2, it does not interact with ERK in yeast two-hybrid assays. HSV-2 Us2 lacks prenylation signals and other canonical membrane-targeting motifs yet is tightly associated with detergent-insoluble membranes and localizes predominantly to recycling endosomes. Experiments to identify cellular proteins that facilitate HSV-2 Us2 membrane association were inconclusive; however, these studies led to the identification of HSV-2 Us2 as a ubiquitin-interacting protein, providing new insight into the functions of HSV-2 Us2.

Integration of lateral porous silicon membranes into planar microfluidics.

PubMed

Leïchlé, Thierry; Bourrier, David

2015-02-07

In this work, we present a novel fabrication process that enables the monolithic integration of lateral porous silicon membranes into single-layer planar microchannels. This fabrication technique relies on the patterning of local electrodes to guide pore formation horizontally within the membrane and on the use of silicon-on-insulator substrates to spatially localize porous silicon within the channel depth. The feasibility of our approach is studied by current flow analysis using the finite element method and supported by creating 10 μm long mesoporous membranes within 20 μm deep microchannels. The fabricated membranes are demonstrated to be potentially useful for dead-end microfiltration by adequately retaining 300 nm diameter beads while macromolecules such as single-stranded DNA and immunoglobulin G permeate the membrane. The experimentally determined fluidic resistance is in accordance with the theoretical value expected from the estimated pore size and porosity. The work presented here is expected to greatly simplify the integration of membranes capable of size exclusion based separation into fluidic devices and opens doors to the use of porous silicon in planar lab on a chip devices.

Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

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

Lazarovici, P.; Yavin, E.

1986-11-04

The pharmacokinetic interaction of an affinity-purified /sup 125/I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10/sup -9/ M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme couldmore » not dissociate the membrane-bound toxin formed at 4 or 37/sup 0/C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4/sup 0/C but not at 37/sup 0/C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin.« less

Nesprin 4 is an outer nuclear membrane protein that can induce kinesin-mediated cell polarization

PubMed Central

Roux, Kyle J.; Crisp, Melissa L.; Liu, Qian; Kim, Daein; Kozlov, Serguei; Stewart, Colin L.; Burke, Brian

2009-01-01

Nucleocytoplasmic coupling is mediated by outer nuclear membrane (ONM) nesprin proteins and inner nuclear membrane Sun proteins. Interactions spanning the perinuclear space create nesprin–Sun complexes connecting the cytoskeleton to nuclear components. A search for proteins displaying a conserved C-terminal sequence present in nesprins 1–3 identified nesprin 4 (Nesp4), a new member of this family. Nesp4 is a kinesin-1-binding protein that displays Sun-dependent localization to the ONM. Expression of Nesp4 is associated with dramatic changes in cellular organization involving relocation of the centrosome and Golgi apparatus relative to the nucleus. These effects can be accounted for entirely by Nesp4's kinesin-binding function. The implication is that Nesp4 may contribute to microtubule-dependent nuclear positioning. PMID:19164528

Properties and degradation of the gasket component of a proton exchange membrane fuel cell--a review.

PubMed

Basuli, Utpal; Jose, Jobin; Lee, Ran Hee; Yoo, Yong Hwan; Jeong, Kwang-Un; Ahn, Jou-Hyeon; Nah, Changwoon

2012-10-01

Proton exchange membrane (PEM) fuel cell stack requires gaskets and seals in each cell to keep the reactant gases within their respective regions. Gasket performance is integral to the successful long-term operation of a fuel cell stack. This review focuses on properties, performance and degradation mechanisms of the different polymer gasket materials used in PEM fuel cell under normal operating conditions. The different degradation mechanisms and their corresponding representative mitigation strategies are also presented here. Summary of various properties of elastomers and their advantages and disadvantages in fuel cell'environment are presented. By considering the level of chemical degradation, mechanical properties and cost effectiveness, it can be proposed that EPDM is one of the best choices for gasket material in PEM fuel cell. Finally, the challenges that remain in using rubber component as in PEM fuel cell, as well as the prospects for exploiting them in the future are discussed.

Focus on membrane differentiation and membrane domains in the prokaryotic cell.

PubMed

Boekema, Egbert J; Scheffers, Dirk-Jan; van Bezouwen, Laura S; Bolhuis, Henk; Folea, I Mihaela

2013-01-01

. More recently, light microscopy in combination with the use of fluorescent dyes has become an attractive technique for protein localization with the natural membrane. However, the resolution problem in light microscopy remains and overinterpretation of observed phenomena is a pitfall. Thus, light microscopy as a stand-alone technique is not sufficient to prove, for instance, the long-range helical distribution of proteins in membrane such as MinD spirals in Bacillus subtilis. Electron tomography is an emerging electron microscopy technique that can provide three-dimensional reconstructions of small, nonchemically fixed bacteria. It will become a useful tool for studying prokaryotic membranes in more detail and is expected to collect information complementary to those of advanced light microscopy. Together, microscopy techniques can meet the challenge of the coming years: to specify membrane structures in more detail and to bring them to the level of specific protein-protein interactions. Copyright © 2013 S. Karger AG, Basel.

Proteomic mapping of cytosol-facing outer mitochondrial and ER membranes in living human cells by proximity biotinylation

PubMed Central

Hung, Victoria; Lam, Stephanie S; Udeshi, Namrata D; Svinkina, Tanya; Guzman, Gaelen; Mootha, Vamsi K; Carr, Steven A; Ting, Alice Y

2017-01-01

The cytosol-facing membranes of cellular organelles contain proteins that enable signal transduction, regulation of morphology and trafficking, protein import and export, and other specialized processes. Discovery of these proteins by traditional biochemical fractionation can be plagued with contaminants and loss of key components. Using peroxidase-mediated proximity biotinylation, we captured and identified endogenous proteins on the outer mitochondrial membrane (OMM) and endoplasmic reticulum membrane (ERM) of living human fibroblasts. The proteomes of 137 and 634 proteins, respectively, are highly specific and highlight 94 potentially novel mitochondrial or ER proteins. Dataset intersection identified protein candidates potentially localized to mitochondria-ER contact sites. We found that one candidate, the tail-anchored, PDZ-domain-containing OMM protein SYNJ2BP, dramatically increases mitochondrial contacts with rough ER when overexpressed. Immunoprecipitation-mass spectrometry identified ribosome-binding protein 1 (RRBP1) as SYNJ2BP’s ERM binding partner. Our results highlight the power of proximity biotinylation to yield insights into the molecular composition and function of intracellular membranes. DOI: http://dx.doi.org/10.7554/eLife.24463.001 PMID:28441135

Active Nuclear Import of Membrane Proteins Revisited

PubMed Central

Laba, Justyna K.; Steen, Anton; Popken, Petra; Chernova, Alina; Poolman, Bert; Veenhoff, Liesbeth M.

2015-01-01

It is poorly understood how membrane proteins destined for the inner nuclear membrane pass the crowded environment of the Nuclear Pore Complex (NPC). For the Saccharomyces cerevisiae proteins Src1/Heh1 and Heh2, a transport mechanism was proposed where the transmembrane domains diffuse through the membrane while the extralumenal domains encoding a nuclear localization signal (NLS) and intrinsically disordered linker (L) are accompanied by transport factors and travel through the NPC. Here, we validate the proposed mechanism and explore and discuss alternative interpretations of the data. First, to disprove an interpretation where the membrane proteins become membrane embedded only after nuclear import, we present biochemical and localization data to support that the previously used, as well as newly designed reporter proteins are membrane-embedded irrespective of the presence of the sorting signals, the specific transmembrane domain (multipass or tail anchored), independent of GET, and also under conditions that the proteins are trapped in the NPC. Second, using the recently established size limit for passive diffusion of membrane proteins in yeast, and using an improved assay, we confirm active import of polytopic membrane protein with extralumenal soluble domains larger than those that can pass by diffusion on similar timescales. This reinforces that NLS-L dependent active transport is distinct from passive diffusion. Thirdly, we revisit the proposed route through the center of the NPC and conclude that the previously used trapping assay is, unfortunately, poorly suited to address the route through the NPC, and the route thus remains unresolved. Apart from the uncertainty about the route through the NPC, the data confirm active, transport factor dependent, nuclear transport of membrane-embedded mono- and polytopic membrane proteins in baker’s yeast. PMID:26473931

Imaging plasma membrane deformations with pTIRFM.

PubMed

Passmore, Daniel R; Rao, Tejeshwar C; Peleman, Andrew R; Anantharam, Arun

2014-04-02

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress. Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or diD. DiD intercalates in the membrane plane with a "fixed" orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561 nm laser (p-pol, s-pol). A 488 nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Study on corrosion migrations within catalyst-coated membranes of proton exchange membrane electrolyzer cells

DOE PAGES

Mo, Jingke; Steen, Stuart; Kang, Zhenye; ...

2017-10-09

The corrosion of low-cost, easily manufactured metallic components inside the electrochemical environment of proton exchange membrane electrolyzer cells (PEMECs) has a significant effect on their performance and durability. Here, 316 stainless steel (SS) mesh was used as a model liquid/gas diffusion layer material to investigate the migration of corrosion products in the catalyst-coated membrane of a PEMEC. Iron and nickel cation particles were found distributed throughout the anode catalyst layer, proton exchange membrane, and cathode catalyst layer, as revealed by scanning transmission electron microscopy and energy dispersive X-ray spectroscopy. Our results indicate the corrosion products of 316 SS are transportedmore » from anode to cathode through the nanochannels of the Nafion membrane, resulting in impeded proton transport and overall PEMEC performance loss.« less

Study on corrosion migrations within catalyst-coated membranes of proton exchange membrane electrolyzer cells

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

Mo, Jingke; Steen, Stuart; Kang, Zhenye

The corrosion of low-cost, easily manufactured metallic components inside the electrochemical environment of proton exchange membrane electrolyzer cells (PEMECs) has a significant effect on their performance and durability. Here, 316 stainless steel (SS) mesh was used as a model liquid/gas diffusion layer material to investigate the migration of corrosion products in the catalyst-coated membrane of a PEMEC. Iron and nickel cation particles were found distributed throughout the anode catalyst layer, proton exchange membrane, and cathode catalyst layer, as revealed by scanning transmission electron microscopy and energy dispersive X-ray spectroscopy. Our results indicate the corrosion products of 316 SS are transportedmore » from anode to cathode through the nanochannels of the Nafion membrane, resulting in impeded proton transport and overall PEMEC performance loss.« less

Membranes, mechanics, and intracellular transport

NASA Astrophysics Data System (ADS)

Parthasarathy, Raghuveer

2012-10-01

Cellular membranes are remarkable materials -- self-assembled, flexible, two-dimensional fluids. Understanding how proteins manipulate membrane curvature is crucial to understanding the transport of cargo in cells, yet the mechanical activities of trafficking proteins remain poorly understood. Using an optical-trap based assay involving dynamic deformation of biomimetic membranes, we have examined the behavior of Sar1, a key component of the COPII family of transport proteins. We find that Sar1 from yeast (S. cerevisiae) lowers membrane rigidity by up to 100% as a function of its concentration, thereby lowering the energetic cost of membrane deformation. Human Sar1 proteins can also lower the mechanical rigidity of the membranes to which they bind. However, unlike the yeast proteins, the rigidity is not a monotonically decreasing function of concentration but rather shows increased rigidity and decreased mobility at high concentrations that implies interactions between proteins. In addition to describing this study of membrane mechanics, I'll also discuss some topics relevant to a range of biophysical investigations, such as the insights provided by imaging methods and open questions in the dynamics of multicellular systems.